Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 1 FAK inhibition reduces Skp2 expression by promoting nuclear localization of FAK. FAK inhibitor (VS-4718) was used at 2.5 mM for the indicated times. (A, C, D, and F) Representative blots (n = 3). (A) Skp2 levels reduced upon pharmacological FAK inhibition monitored by immunoblotting with mouse VSMC lysates. Skp2 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. (B) Mouse VSMCs were treated with FAK inhibitor for 2 days, and Skp2 mRNA levels were measured via RT-qPCR (± SEM, n = 6, Student t-test). (C) Human coronary artery SMCs (hCASMCs) were treated with FAK inhibitor. Skp2, p27, and p21 blots were quantified relative to GAPDH. pY397 FAK was quantified relative to total FAK. (D) Skp2 lev- els in FAK-WT and FAK-KD mouse VSMCs. Skp2 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. (E) Skp2 mRNA levels were measured in FAK-WT and FAK-KD mouse VSMCs via RT-qPCR (± SEM, n = 6, Student t-test). (F) Mouse VSMCs were treated with FAK inhibitor with or without proteasome inhibitor (MG132, 20mM) for 6 h. Skp2 blots were quantified relative to actin. pY397 FAK was quantified relative to total FAK. (G) Mouse VSMCs were treated with or without FAK inhibitor for 24h. FAK, Skp2, pY397 FAK, p27, and p21 immunostainings are shown. PCNA was used as a proliferation marker. Representative images (n = 4). Scale bar: 20mm.

Article Snippet: Two different shRNAs targeting mouse Skp2 were cloned into pSicoR-mCherry (Addgene #21907) using XhoI and HpaI restriction site. shRNA sequences for Skp2-1 and -2 are listed in Online Table I.

Techniques: Inhibition, Expressing, Western Blot, Quantitative RT-PCR, Marker

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 2 FAK interacts with Skp2 and the APC/C E3 ligase activator Fzr1 through FAK FERM domain. FAK inhibitor (VS-4718) was used at 2.5 mM for the indicated times. (A–E) Representative blots (n = 3). (A) FAK interacts with Skp2 and Fzr1 by immunoprecipitation (IP) with VSMC lysates. Mouse VSMCs were treated with FAK inhibitor together with proteasome inhibitor (MG132, 20lM) for 6 h. FAK and Fzr1 blots within Skp2-IP were quantified relative to untreated controls. The arrow head indicates the heavy chain of IgG. (B) Immunoblots of mouse VSMC cytosolic (C) and nuclear (N) fractionated lysates for FAK, Fzr1, and Skp2. PARP and GAPDH as nuclear and cytosolic markers. Cytosolic or nuclear FAK, Skp2, and Fzr1 blots were quantified relative to GAPDH or PARP.. (C) FAK FERM binds to both Skp2 and Fzr1 in 293T cells transfected with GFP-fused FAK and its subdomains. Skp2 and Fzr1 blots were quantified relative to GFP. (D) FAK FERM F1 lobe binds Skp2 and F3 lobe binds Fzr1 in 293T cells co-transfected with Myc-Skp2, HA-Fzr1, and FAK FERM F1, F2, and F3 lobes as GST fusion proteins. Skp2 and Fzr1 blots were quantified relative to GST. (E) Mouse VSMCs were treated with FAK inhibitor. pY397 FAK and Fzr1 blots were quantified relative to GAPDH. (F) Fzr1 mRNA levels were measured in mouse VSMCs treated with FAK inhibitor for 2 days or in genetic FAK inhibition via RT-qPCR (± SEM, n = 6, Student t-test). (G) Nuclear localization of FAK plays a key role in Fzr1 and Skp2 degradation. FAK-/-

Article Snippet: Two different shRNAs targeting mouse Skp2 were cloned into pSicoR-mCherry (Addgene #21907) using XhoI and HpaI restriction site. shRNA sequences for Skp2-1 and -2 are listed in Online Table I.

Techniques: Immunoprecipitation, Western Blot, Transfection, Inhibition, Quantitative RT-PCR

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 3 Skp2 is increased after wire injury and pharmacological FAK catalytic inhibition significantly reduces Skp2 and neointimal hyperplasia. Mice were treated with vehicle or VS-4718 (50 mg/kg) twice daily following wire injury. Representative H&E staining of femoral artery cross sections 14 days after wire injury for vehicle or VS-4718-treated (A, n = 5), Scale bar: 50mm. Intima/media (I/M) ratios were quantified (± SD, n = 5, **P < 0.005 vs. vehicle injury, two- way ANOVA followed by Sidak multiple comparisons test). (B) Representative immunoblots of femoral arteries 14days postinjury for pY397 FAK, FAK, Skp2, p27, p21, and GAPDH as loading control. Skp2, p27, and p21 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. Representative blots (n= 4). (C) Representative immunofluorescence staining of femoral arteries 14 days postinjury for pY397 FAK, FAK, p27, p21, Skp2, Fzr1, and a-SMA. Red, green, and blue (DAPI) were merged (n = 5). Dotted line in image marks the external or internal elastic lamina. White line shows endothelial layer. Scale bar: 20mm.

Article Snippet: Two different shRNAs targeting mouse Skp2 were cloned into pSicoR-mCherry (Addgene #21907) using XhoI and HpaI restriction site. shRNA sequences for Skp2-1 and -2 are listed in Online Table I.

Techniques: Inhibition, Staining, Western Blot, Control, Immunofluorescence

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 4 VSMC-specific FAK-KD mice development reduces hyperplasia and exhibits less Skp2 and more p27, p21 expression after wire injury. (A) Representative H&E staining of femoral artery cross sections 2 weeks after wire injury for genetic FAK-WT or FAK-KD mice (n = 5). Scale bar: 50mm. Intima/media (I/M) ratios were quantified (± SD, n = 5, **P< 0.005 vs. FAK-WT injury, two-way ANOVA followed by Sidak multiple comparisons test). (B) Representative immunoblots of femoral arteries 14days postinjury for pY397 FAK, FAK, Skp2, p27, p21 GAPDH as loading control. Skp2, p27, and p21 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. Representative blots (n = 4). (C) Representative immunofluores- cence staining of FAK-WT and FAK-KD femoral arteries 2 weeks postinjury for pY397 FAK, FAK, p27, p21, Skp2, Fzr1, and a-SMA. Red, green, and blue (DAPI) were merged (n = 4). Dotted line in image marks the external or internal elastic lamina. White line shows endothelial layer. Scale bar: 20mm.

Article Snippet: Two different shRNAs targeting mouse Skp2 were cloned into pSicoR-mCherry (Addgene #21907) using XhoI and HpaI restriction site. shRNA sequences for Skp2-1 and -2 are listed in Online Table I.

Techniques: Expressing, Staining, Western Blot, Control

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 5 Skp2 directly regulates CDKI expression and functions independently of cyclin D1 in the cell cycle progression. (A) Mouse VSMCs were trans- duced to overexpress Skp2. Skp2, p27, and p21 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. Representative blots (n = 3). (B) Skp2 overexpression slightly increased mouse VSMC proliferation (± SD, n = 3, *P < 0.01, **P < 0.005 vs. control, two-way ANOVA followed by Sidak multiple comparisons test). (C) p27 or p21 mRNA levels were measured in mouse VSMCs overexpressing Skp2 via RT-qPCR (± SEM, n = 3, paired t-test). (D) shRNA-mediated knockdown of Skp2 increased p27 and p21 protein. Skp2, p27, and p21 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. Representative blots (n = 3). (E) Skp2, p27, and p21 mRNA levels were measured in Skp2 knockdown mouse VSMCs via RT-qPCR (± SEM, n = 3, **P < 0.005 vs. scramble, Student t-test). (F) Skp2 knockdown reduced mouse VSMC proliferation (± SD, n = 3, **P < 0.005 vs. scramble, two-way ANOVA followed by Sidak multiple comparisons test). (G) Cyclin D1 and Skp2 were overexpressed in FAK-WT and FAK-KD mouse VSMCs. Skp2, cyclin D1, p27, and p21 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. Representative blots (n = 3). (H) Skp2 overexpression rescues defect of mouse VSMCs proliferation in cyclin D1-overexpressing FAK-KD VSMCs (± SD, n = 3, **P < 0.005 vs. FAK-KD, two-way ANOVA followed by Sidak multiple comparisons test).

Article Snippet: Two different shRNAs targeting mouse Skp2 were cloned into pSicoR-mCherry (Addgene #21907) using XhoI and HpaI restriction site. shRNA sequences for Skp2-1 and -2 are listed in Online Table I.

Techniques: Expressing, Over Expression, Control, Quantitative RT-PCR, shRNA, Knockdown

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 6 Knockdown of Skp2 reduces wire injury-induced neointi- mal hyperplasia. Femoral arteries were coated with either lentivirus encoding mCherry , scramble shRNA (shScr), or Skp2 shRNA (shSkp2) immediately following wire injury. After 2 week wire injury, immunos- tainings for a-SMA, Skp2, and p27 were shown. SMA (green), mCherry (red), and DAPI (blue) were merged (n = 4). mCherry was used to ver- ify viral infection. Dotted line in image marks the external or internal elastic lamina. White line shows endothelial layer. Scale bar: 20mm.

Article Snippet: Two different shRNAs targeting mouse Skp2 were cloned into pSicoR-mCherry (Addgene #21907) using XhoI and HpaI restriction site. shRNA sequences for Skp2-1 and -2 are listed in Online Table I.

Techniques: Knockdown, shRNA, Infection

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 7 FAK inhibition blocks neointimal hyperplasia in overex- pressing Skp2 mice upon wire injury. Femoral arteries were coated with Myc-Skp2 overexpressing lentivirus immediately following wire in- jury. Mice were treated with vehicle or VS-4718 (50 mg/kg) twice daily. After 2 week wire injury, immunostainings for a-SMA, Myc, FAK, pY397 FAK, p27, and Skp2 were shown. SMA (green), Myc-Skp2 or pY397 (red), and DAPI (blue) were merged (n = 4). Dotted line in image marks the external or internal elastic lamina. White line shows endothelial layer. Scale bar: 20mm.

Article Snippet: Two different shRNAs targeting mouse Skp2 were cloned into pSicoR-mCherry (Addgene #21907) using XhoI and HpaI restriction site. shRNA sequences for Skp2-1 and -2 are listed in Online Table I.

Techniques: Inhibition

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a ) FBXW2 binds to SKP2 in vivo : Lysates from H1299 cells were pulled down with anti-FBXW2 (left panel) or anti-SKP2 (right panel), followed by IB. ( b ) FBXW2 failed to bind to a SKP2 mutant: H1299 cells were tranfected with indicated plasmids, followed by FLAG IP and HA IB or direct IB. ( c , d ) Ectopically expressed FBXW2 reduces the endogenous levels of SKP2 protein, but not mRNA: Cells were transfected with HA-FBXW2, followed by IB ( c ) or qRT-PCR for SKP2 ( d ). Error bars indicate mean +s.d. of three repeats. ( e , f ) FBXW2 overexpression decreases the levels of the exogenous and endogenous SKP2 proteins: Cells were co-transfected with indicated plasmids, followed by IB 48 h post transfection. ( g ) FBXW2 depletion increases the levels of SKP2 protein, but not mRNA: Cells were transfected with FBXW2 siRNA and scramble siRNA, followed by IB or qRT-PCR for SKP2. Error bars indicate mean+s.d. of three repeats. ( h ) FBXW2-induced SKP2 degradation is independent of CDH1: Cells were transfected with FLAG-FBXW2 and/or siRNA against CDH1, and then harvested for IB. ( i ) FBXW2 shortens SKP2 half-life: Cells were transfected with HA-FBXW2, and switched 48 h post transfection to fresh medium containing CHX for indicated periods with or without MG132 treatment for last 2 h, and then harvested for IB. The band density was quantified. ( j ) FBXW2 knockdown extends SKP2 half-life: Cells were transfected with siRNA targeting FBXW2. Cells were switched 48 h later to fresh medium containing CHX for indicated periods and harvested for IB. The band density was quantified. ( k , l ) FBXW2 promotes ubiquitylation of SKP2 ( k ), but not SKP2-3A mutant ( l ): Cells were transfected with indicated plasmids, followed by Ni-beads pull-down and IB for SKP2. ( m ) FBXW2 promotes SKP2 ubiquitylation by in vitro assay: H1299 cells were transfected with indicated plasmids. Pull-down purified FBXW2 and FBXW2ΔF (E3s), Pull-down purified SKP2 (substrate), were added into a reaction mixture containing ATP, ubiquitin, E1 and E2, followed by IB using anti-FLAG Ab. ( n ) FBXW2 promotes SKP2 ubiquitylation via K48 linkage: Cells were transfected with indicated plasmids, lysed under denatured condition at 6 M guanidinium solution, followed by Ni-beads pull-down and IB for SKP2.

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, Cellsignal; 1:500 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:1,000 overnight, 4 °C); mouse-ki-67 (#550609, BD Pharmingen; 1:1,000 overnight, 4 °C); rabbit-Cleaved Caspase-3 (Asp175) (#9661, Cellsignal; 1:500 overnight, 4 °C).

Techniques: In Vivo, Mutagenesis, Transfection, Quantitative RT-PCR, Over Expression, Knockdown, In Vitro, Purification, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a ) Fluctuation of the levels of F-box proteins and VRK2 kinase during cell cycle progression: H358 cells were serum starved for 48 h, followed by serum addition. Cells were harvested at indicated time points and subjected to FACS and IB analyses using indicated Abs. ( b – f ) FBXW2-3A mutant rescues growth-promoting phenotype induced by β-TrCP1 overexpression ( b – d ), and wt FBXW2 rescues growth-promoting phenotype induced by SKP2 overexpression ( b , e , f ): H1299 cells were co-transfected with the indicated plasmids, followed by IB ( b ), ATP-lite proliferation assay ( n =3) ( c , e ) and clonogenic survival assay ( n =3) ( d , f ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01. ( g – i ) FBXW2 depletion stimulates cell growth, which is abrogated by simultaneous SKP2 depletion: H1299 cells were transfected with shRNAs targeting FBXW2 alone or in combination with shRNA targeting SKP2, along with scramble control, and then harvested for IB ( g ), ATP-lite proliferation assay ( n =3) ( h ) and clonogenic survival assay ( n =3) ( i ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01; *** P <0.001. ( j , k ) FBXW2 depletion stimulates tumour growth, which is abrogated by simultaneous SKP2 depletion: H1299 cells were transfected with shRNAs targeting FBXW2 alone or in combination with shRNA targeting SKP2, along with scramble control, followed by injection (1 × 10 6 cells) into nude mice. Tumour growth was observed for 33 days ( j ). Tumours were then harvested, photographed and weighted ( k ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, Cellsignal; 1:500 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:1,000 overnight, 4 °C); mouse-ki-67 (#550609, BD Pharmingen; 1:1,000 overnight, 4 °C); rabbit-Cleaved Caspase-3 (Asp175) (#9661, Cellsignal; 1:500 overnight, 4 °C).

Techniques: Mutagenesis, Over Expression, Transfection, Proliferation Assay, Clonogenic Cell Survival Assay, shRNA, Control, Injection

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a – c ) Expression levels among three F-box proteins in lung cancer cell lines and tissues: Cell lysates from eight lung cancer cell lines and one immortalized line (BEAS-2B) were subjected to IB ( a ); SE: Short exposure; LE: Long exposure. Lung cancer tissue microarrays were stained with indicated Abs and photographed ( b , Scale bars, 100 μm), and data were then analysed using SPSS software to obtain coefficient ( c ; P <0.001, Pearson's test). ( d – f ) Protein expression of three F-box proteins in lung cancer and their relationship with patient survival: Continuous protein expression values were classified into the low and high groups with equal number of patients, and 5-year survival time was used for Kaplan-Meier survival analysis ( d – f ). Kaplan-Meier survival analysis indicated that patient with higher expression of FBXW2 was related to a better overall survival (log-rank test, P =0.032) ( e ); Higher expression of β-TrCP1 and SKP2 were related to a worse overall patient survival (log-rank test, P <0.001 and 0.001, respectively) ( d , f ).

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, Cellsignal; 1:500 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:1,000 overnight, 4 °C); mouse-ki-67 (#550609, BD Pharmingen; 1:1,000 overnight, 4 °C); rabbit-Cleaved Caspase-3 (Asp175) (#9661, Cellsignal; 1:500 overnight, 4 °C).

Techniques: Expressing, Staining, Software

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a ) FBXW2 mutants have reduced binding with SCF components: H1299 cells were transfected with indicated plasmids, followed by G418 selection. Resistant clones were pooled and subjected to FLAG-bead IP and IB with indicated Abs. ( b , c ) FBXW2 mutants MU-S84C ( b ) and MU-E269K ( c ) are unable to shorten SKP2 protein half-life: Stable H1299 cells were incubated with CHX for indicated time periods and harvested for IB. The band density was quantified. ( d , e ) Loss- or gain-of-function of FBXW2 mutants: H1299 cells stably expressing MU-S84C and MU-E269K mutants were subjected to ATP-lite proliferation assay ( n =3) ( d ), and clonogenic survival assay ( n =3) ( e ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01; *** P <0.001. ( f , g ) FBXW2 mutants either lose the tumour suppressor function or gain the oncogenic function in vivo : H1299 cells stably expressing MU-S84C and MU-E269K mutants (1 × 10 6 cells) were inoculated s.c. in both flanks of nude mice. The tumour growth was monitored twice a week for up to 28 days and growth curve plotted ( f ). Tumour tissues were harvested, photographed and weighed at 28 days ( g ). Student's t -test was used to compare each experimental group with the control group. Shown are mean±s.e.m., * P <0.05; ** P <0.01; *** P <0.001. ( h ) Immunohistochemical staining of xenograft tumour tissues. Tumour tissues from four groups of mice were fixed, sectioned and stained with indicated antibodies. Scale bars: 100 μm. Shown are mean±s.e.m., * P <0.05; ** P <0.01. ( i ) The oncogene-tumour suppressor-oncogene axis—a working model: During tumorigenesis, oncogenic β-TrCP1 is activated to promote ubiquitylation and degradation of FBXW2 tumour suppressor, resulting in abrogation of FBXW2-induced SKP2 degradation. Accumulated oncogenic SKP2 promotes ubiquitylation and degradation of tumour suppressors such as p21, p27, p130, FOXO1, and leading to activation of CDKs, E2F and mTOR, eventually to uncontrolled proliferation of cancer cells.

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, Cellsignal; 1:500 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:1,000 overnight, 4 °C); mouse-ki-67 (#550609, BD Pharmingen; 1:1,000 overnight, 4 °C); rabbit-Cleaved Caspase-3 (Asp175) (#9661, Cellsignal; 1:500 overnight, 4 °C).

Techniques: Binding Assay, Transfection, Selection, Clone Assay, Incubation, Stable Transfection, Expressing, Proliferation Assay, Clonogenic Cell Survival Assay, In Vivo, Control, Immunohistochemical staining, Staining, Activation Assay

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 1 FAK inhibition reduces Skp2 expression by promoting nuclear localization of FAK. FAK inhibitor (VS-4718) was used at 2.5 mM for the indicated times. (A, C, D, and F) Representative blots (n = 3). (A) Skp2 levels reduced upon pharmacological FAK inhibition monitored by immunoblotting with mouse VSMC lysates. Skp2 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. (B) Mouse VSMCs were treated with FAK inhibitor for 2 days, and Skp2 mRNA levels were measured via RT-qPCR (± SEM, n = 6, Student t-test). (C) Human coronary artery SMCs (hCASMCs) were treated with FAK inhibitor. Skp2, p27, and p21 blots were quantified relative to GAPDH. pY397 FAK was quantified relative to total FAK. (D) Skp2 lev- els in FAK-WT and FAK-KD mouse VSMCs. Skp2 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. (E) Skp2 mRNA levels were measured in FAK-WT and FAK-KD mouse VSMCs via RT-qPCR (± SEM, n = 6, Student t-test). (F) Mouse VSMCs were treated with FAK inhibitor with or without proteasome inhibitor (MG132, 20mM) for 6 h. Skp2 blots were quantified relative to actin. pY397 FAK was quantified relative to total FAK. (G) Mouse VSMCs were treated with or without FAK inhibitor for 24h. FAK, Skp2, pY397 FAK, p27, and p21 immunostainings are shown. PCNA was used as a proliferation marker. Representative images (n = 4). Scale bar: 20mm.

Article Snippet: GFP-tagged FAK WT, FERM, kinase, and C-terminal domain plasmid were used as previously described.34,35 FAK FERM F1 (33-132), FERM F2 (128-253), and FERM F3 (253-353) lobes were amplified by PCR and cloned into the pEBG mammalian GST fusion expression vector as described.35 Myc-tagged Skp2 (#19947) and HA-tagged Fzr1 (#11596) plasmid constructs were from Addgene.

Techniques: Inhibition, Expressing, Western Blot, Quantitative RT-PCR, Marker

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 2 FAK interacts with Skp2 and the APC/C E3 ligase activator Fzr1 through FAK FERM domain. FAK inhibitor (VS-4718) was used at 2.5 mM for the indicated times. (A–E) Representative blots (n = 3). (A) FAK interacts with Skp2 and Fzr1 by immunoprecipitation (IP) with VSMC lysates. Mouse VSMCs were treated with FAK inhibitor together with proteasome inhibitor (MG132, 20lM) for 6 h. FAK and Fzr1 blots within Skp2-IP were quantified relative to untreated controls. The arrow head indicates the heavy chain of IgG. (B) Immunoblots of mouse VSMC cytosolic (C) and nuclear (N) fractionated lysates for FAK, Fzr1, and Skp2. PARP and GAPDH as nuclear and cytosolic markers. Cytosolic or nuclear FAK, Skp2, and Fzr1 blots were quantified relative to GAPDH or PARP.. (C) FAK FERM binds to both Skp2 and Fzr1 in 293T cells transfected with GFP-fused FAK and its subdomains. Skp2 and Fzr1 blots were quantified relative to GFP. (D) FAK FERM F1 lobe binds Skp2 and F3 lobe binds Fzr1 in 293T cells co-transfected with Myc-Skp2, HA-Fzr1, and FAK FERM F1, F2, and F3 lobes as GST fusion proteins. Skp2 and Fzr1 blots were quantified relative to GST. (E) Mouse VSMCs were treated with FAK inhibitor. pY397 FAK and Fzr1 blots were quantified relative to GAPDH. (F) Fzr1 mRNA levels were measured in mouse VSMCs treated with FAK inhibitor for 2 days or in genetic FAK inhibition via RT-qPCR (± SEM, n = 6, Student t-test). (G) Nuclear localization of FAK plays a key role in Fzr1 and Skp2 degradation. FAK-/-

Article Snippet: GFP-tagged FAK WT, FERM, kinase, and C-terminal domain plasmid were used as previously described.34,35 FAK FERM F1 (33-132), FERM F2 (128-253), and FERM F3 (253-353) lobes were amplified by PCR and cloned into the pEBG mammalian GST fusion expression vector as described.35 Myc-tagged Skp2 (#19947) and HA-tagged Fzr1 (#11596) plasmid constructs were from Addgene.

Techniques: Immunoprecipitation, Western Blot, Transfection, Inhibition, Quantitative RT-PCR

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 3 Skp2 is increased after wire injury and pharmacological FAK catalytic inhibition significantly reduces Skp2 and neointimal hyperplasia. Mice were treated with vehicle or VS-4718 (50 mg/kg) twice daily following wire injury. Representative H&E staining of femoral artery cross sections 14 days after wire injury for vehicle or VS-4718-treated (A, n = 5), Scale bar: 50mm. Intima/media (I/M) ratios were quantified (± SD, n = 5, **P < 0.005 vs. vehicle injury, two- way ANOVA followed by Sidak multiple comparisons test). (B) Representative immunoblots of femoral arteries 14days postinjury for pY397 FAK, FAK, Skp2, p27, p21, and GAPDH as loading control. Skp2, p27, and p21 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. Representative blots (n= 4). (C) Representative immunofluorescence staining of femoral arteries 14 days postinjury for pY397 FAK, FAK, p27, p21, Skp2, Fzr1, and a-SMA. Red, green, and blue (DAPI) were merged (n = 5). Dotted line in image marks the external or internal elastic lamina. White line shows endothelial layer. Scale bar: 20mm.

Article Snippet: GFP-tagged FAK WT, FERM, kinase, and C-terminal domain plasmid were used as previously described.34,35 FAK FERM F1 (33-132), FERM F2 (128-253), and FERM F3 (253-353) lobes were amplified by PCR and cloned into the pEBG mammalian GST fusion expression vector as described.35 Myc-tagged Skp2 (#19947) and HA-tagged Fzr1 (#11596) plasmid constructs were from Addgene.

Techniques: Inhibition, Staining, Western Blot, Control, Immunofluorescence

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 4 VSMC-specific FAK-KD mice development reduces hyperplasia and exhibits less Skp2 and more p27, p21 expression after wire injury. (A) Representative H&E staining of femoral artery cross sections 2 weeks after wire injury for genetic FAK-WT or FAK-KD mice (n = 5). Scale bar: 50mm. Intima/media (I/M) ratios were quantified (± SD, n = 5, **P< 0.005 vs. FAK-WT injury, two-way ANOVA followed by Sidak multiple comparisons test). (B) Representative immunoblots of femoral arteries 14days postinjury for pY397 FAK, FAK, Skp2, p27, p21 GAPDH as loading control. Skp2, p27, and p21 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. Representative blots (n = 4). (C) Representative immunofluores- cence staining of FAK-WT and FAK-KD femoral arteries 2 weeks postinjury for pY397 FAK, FAK, p27, p21, Skp2, Fzr1, and a-SMA. Red, green, and blue (DAPI) were merged (n = 4). Dotted line in image marks the external or internal elastic lamina. White line shows endothelial layer. Scale bar: 20mm.

Article Snippet: GFP-tagged FAK WT, FERM, kinase, and C-terminal domain plasmid were used as previously described.34,35 FAK FERM F1 (33-132), FERM F2 (128-253), and FERM F3 (253-353) lobes were amplified by PCR and cloned into the pEBG mammalian GST fusion expression vector as described.35 Myc-tagged Skp2 (#19947) and HA-tagged Fzr1 (#11596) plasmid constructs were from Addgene.

Techniques: Expressing, Staining, Western Blot, Control

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 5 Skp2 directly regulates CDKI expression and functions independently of cyclin D1 in the cell cycle progression. (A) Mouse VSMCs were trans- duced to overexpress Skp2. Skp2, p27, and p21 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. Representative blots (n = 3). (B) Skp2 overexpression slightly increased mouse VSMC proliferation (± SD, n = 3, *P < 0.01, **P < 0.005 vs. control, two-way ANOVA followed by Sidak multiple comparisons test). (C) p27 or p21 mRNA levels were measured in mouse VSMCs overexpressing Skp2 via RT-qPCR (± SEM, n = 3, paired t-test). (D) shRNA-mediated knockdown of Skp2 increased p27 and p21 protein. Skp2, p27, and p21 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. Representative blots (n = 3). (E) Skp2, p27, and p21 mRNA levels were measured in Skp2 knockdown mouse VSMCs via RT-qPCR (± SEM, n = 3, **P < 0.005 vs. scramble, Student t-test). (F) Skp2 knockdown reduced mouse VSMC proliferation (± SD, n = 3, **P < 0.005 vs. scramble, two-way ANOVA followed by Sidak multiple comparisons test). (G) Cyclin D1 and Skp2 were overexpressed in FAK-WT and FAK-KD mouse VSMCs. Skp2, cyclin D1, p27, and p21 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. Representative blots (n = 3). (H) Skp2 overexpression rescues defect of mouse VSMCs proliferation in cyclin D1-overexpressing FAK-KD VSMCs (± SD, n = 3, **P < 0.005 vs. FAK-KD, two-way ANOVA followed by Sidak multiple comparisons test).

Article Snippet: GFP-tagged FAK WT, FERM, kinase, and C-terminal domain plasmid were used as previously described.34,35 FAK FERM F1 (33-132), FERM F2 (128-253), and FERM F3 (253-353) lobes were amplified by PCR and cloned into the pEBG mammalian GST fusion expression vector as described.35 Myc-tagged Skp2 (#19947) and HA-tagged Fzr1 (#11596) plasmid constructs were from Addgene.

Techniques: Expressing, Over Expression, Control, Quantitative RT-PCR, shRNA, Knockdown

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 6 Knockdown of Skp2 reduces wire injury-induced neointi- mal hyperplasia. Femoral arteries were coated with either lentivirus encoding mCherry , scramble shRNA (shScr), or Skp2 shRNA (shSkp2) immediately following wire injury. After 2 week wire injury, immunos- tainings for a-SMA, Skp2, and p27 were shown. SMA (green), mCherry (red), and DAPI (blue) were merged (n = 4). mCherry was used to ver- ify viral infection. Dotted line in image marks the external or internal elastic lamina. White line shows endothelial layer. Scale bar: 20mm.

Article Snippet: GFP-tagged FAK WT, FERM, kinase, and C-terminal domain plasmid were used as previously described.34,35 FAK FERM F1 (33-132), FERM F2 (128-253), and FERM F3 (253-353) lobes were amplified by PCR and cloned into the pEBG mammalian GST fusion expression vector as described.35 Myc-tagged Skp2 (#19947) and HA-tagged Fzr1 (#11596) plasmid constructs were from Addgene.

Techniques: Knockdown, shRNA, Infection

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 7 FAK inhibition blocks neointimal hyperplasia in overex- pressing Skp2 mice upon wire injury. Femoral arteries were coated with Myc-Skp2 overexpressing lentivirus immediately following wire in- jury. Mice were treated with vehicle or VS-4718 (50 mg/kg) twice daily. After 2 week wire injury, immunostainings for a-SMA, Myc, FAK, pY397 FAK, p27, and Skp2 were shown. SMA (green), Myc-Skp2 or pY397 (red), and DAPI (blue) were merged (n = 4). Dotted line in image marks the external or internal elastic lamina. White line shows endothelial layer. Scale bar: 20mm.

Article Snippet: GFP-tagged FAK WT, FERM, kinase, and C-terminal domain plasmid were used as previously described.34,35 FAK FERM F1 (33-132), FERM F2 (128-253), and FERM F3 (253-353) lobes were amplified by PCR and cloned into the pEBG mammalian GST fusion expression vector as described.35 Myc-tagged Skp2 (#19947) and HA-tagged Fzr1 (#11596) plasmid constructs were from Addgene.

Techniques: Inhibition

Journal: iScience

Article Title: A nonautophagic role of ATG5 in regulating cell growth by targeting c-Myc for proteasome-mediated degradation

doi: 10.1016/j.isci.2021.103296

Figure Lengend Snippet:

Article Snippet: Antibodies against ATG5 (mouse), USP28, SKP2, TRIM32, CHIP, and the His tag were purchased from Proteintech (China).

Techniques: Virus, shRNA, Recombinant, Plasmid Preparation, Software, Luciferase, Reporter Gene Assay, Bicinchoninic Acid Protein Assay, Reverse Transcription

Journal: Molecular and cellular endocrinology

Article Title: Melanoma antigen-A11 regulates substrate-specificity of Skp2-mediated protein degradation

doi: 10.1016/j.mce.2016.10.006

Figure Lengend Snippet: [A] HeLa-AR1C-PSA-Luc-A6 (HeLa-AR), LNCaP, CWR-R1, LAPC-4, CV1 and COS1 cells were incubated for 48 h with or without 10 nM DHT in phenol red-free medium containing 10% charcoal stripped serum. Cell extracts (30 μg protein/lane) were probed on the immunoblot using AR, Skp2 and β-actin antibodies. [B] Effects of lentivirus shRNA knockdown of MAGE-A11 and AR on MAGE-A11, Skp2 and AR levels were determined in LAPC-4 cells transduced without virus (-) or with lentivirus expressing MAGE-A11 shRNA-169, 827, 947 or 964, AR shRNA-5, nonspecific empty vector shRNA (NS1) or nonspecific 18-bp shRNA (NS2) as described in Methods. Cells were incubated for 48 h in serum-free medium containing 50 nM DHT and 0.1 μg/ml EGF. Cell extracts (0.1 mg protein/lane) were probed on immunoblots using AR, MAGE1, Skp2 and β-actin antibodies. [C] Effects of lentivirus shRNA knockdown of MAGE-A11 on CWR-R1 cell growth were determined in the absence (dashed lines) or presence of 0.1 nM DHT and 10 ng/ml EGF (solid lines). Cells were selected using puromycin as described in Methods for lentivirus shRNA expression of NS1, NS2, MAGE-A11 shRNA-827 (M827) that did not decrease MAGE-A11 levels, or MAGE-A11 shRNA-947 (M947) or 169 (M169) that decreased MAGE-A11 to different extents. Cell growth assays were performed in triplicate over 4 days and show the mean and S.E.

Article Snippet: Antibodies used to probe the immunoblots included rabbit MAGE1 and MAGE2 against baculovirus expressed human FLAG-MAGE-A11 (0.5-10 μg/ml) ( Su et al., 2013 ); rabbit Skp2 antibody 4358 (Cell Signaling Technology, 1:50-200); mouse FLAG-M2 antibody F3165 (Sigma-Aldrich, 1:200-2000); rabbit HA antibody ab9110 (1:500-2000) and mouse β-actin ab6276 (1:5000) from Abcam; mouse monoclonal HA-tag 12ca5 antibody from the University of North Carolina Antibody Core Facility (1-2 μg/ml); rabbit Skp2 antibody sc-7164 (1:200), rabbit cyclin A sc-751 antibody (1:200), mouse E2F1 antibody sc-193 (1:200), rabbit p107 antibody sc-318 and rabbit p130 antibody sc-317 (1:200) from Santa Cruz; rabbit AR32 (1 μg/ml) ( Quarmby et al., 1990 ) and AR52 antipeptide antibodies (10 μg/ml) ( Lubahn et al., 1988 ).

Techniques: Incubation, Western Blot, shRNA, Knockdown, Virus, Expressing, Plasmid Preparation

Journal: Molecular and cellular endocrinology

Article Title: Melanoma antigen-A11 regulates substrate-specificity of Skp2-mediated protein degradation

doi: 10.1016/j.mce.2016.10.006

Figure Lengend Snippet: [A] Interaction of MAGE-A11 with endogenous Skp2 was shown by expressing 5 μg pCMV-FLAG (-) or pCMV-FLAG-MAGE/10 cm dish of COS1 cells treated for 24 h before harvest with 10 ng/ml EGF in serum-free medium. Four dishes were pooled and cell extracts (60 μg/lane) were analyzed on immunoblots using Skp2 and MAGE2 antibodies. Full-length FLAG-MAGE (lower panel, upper band) was also detected in a smaller form (lower band) that likely resulted from partial proteolysis during extraction. [B] Interaction of MAGE-A11 with endogenous cyclin A was shown by expressing 3 μg pCMV-FLAG (-) or pCMV-FLAG-MAGE/10-cm dish. Immunoprecipitation was performed using 4 pooled 10-cm COS1 cell dishes as described in Methods. Immunoblots of cell extracts (60 μg protein/lane) and immunoprecipitates were probed using cyclin A sc-751 and FLAG-M2 antibodies.

Article Snippet: Antibodies used to probe the immunoblots included rabbit MAGE1 and MAGE2 against baculovirus expressed human FLAG-MAGE-A11 (0.5-10 μg/ml) ( Su et al., 2013 ); rabbit Skp2 antibody 4358 (Cell Signaling Technology, 1:50-200); mouse FLAG-M2 antibody F3165 (Sigma-Aldrich, 1:200-2000); rabbit HA antibody ab9110 (1:500-2000) and mouse β-actin ab6276 (1:5000) from Abcam; mouse monoclonal HA-tag 12ca5 antibody from the University of North Carolina Antibody Core Facility (1-2 μg/ml); rabbit Skp2 antibody sc-7164 (1:200), rabbit cyclin A sc-751 antibody (1:200), mouse E2F1 antibody sc-193 (1:200), rabbit p107 antibody sc-318 and rabbit p130 antibody sc-317 (1:200) from Santa Cruz; rabbit AR32 (1 μg/ml) ( Quarmby et al., 1990 ) and AR52 antipeptide antibodies (10 μg/ml) ( Lubahn et al., 1988 ).

Techniques: Expressing, Western Blot, Extraction, Immunoprecipitation

Journal: Molecular and cellular endocrinology

Article Title: Melanoma antigen-A11 regulates substrate-specificity of Skp2-mediated protein degradation

doi: 10.1016/j.mce.2016.10.006

Figure Lengend Snippet: [A] Sequence alignment of Skp2 amino acid residues 40-60 cyclin A binding region (Ji et al., 2006) with MAGE-A11 amino acid residues 150-170. Homologous amino acid residues are shown in red. [B] Schematic of MAGE-A11 deletion mutants used to test for interaction with cyclin A included MAGEΔ151-217, Δ171-190, Δ221-249 and Δ314-323. Also shown are MAGE-A11 monoubiquitination sites Lys-240 and Lys-245 (Ub-K240 and Ub-K245), checkpoint kinase Chk1 phosphorylation site Thr-360 (Bai and Wilson, 2008), F-box amino acid residues 329-369 (Askew et al., 2009) and MAGE homology domain (MHD, shaded). − and + indicate interaction with cyclin A. [C] The interaction region in MAGE-A11 for cyclin A was shown by immunoprecipitation by expressing 2 μg pcDNA3 (-) or pcDNA3-HA-cyclin A with 2 μg pCMV-FLAG (-), full-length pCMV-FLAG-MAGE or pCMV-FLAG-MAGEΔ151-217, Δ171-190, Δ221-249 or Δ314-323 in COS1 cells. The day after transfection cells were incubated for 18 h in serum-free medium containing 0.1 μg/ml EGF. Immunoblots of cell extracts (50 μg protein/ml) and immunoprecipitates were probed using HA ab9110 and FLAG-M2 antibodies.

Article Snippet: Antibodies used to probe the immunoblots included rabbit MAGE1 and MAGE2 against baculovirus expressed human FLAG-MAGE-A11 (0.5-10 μg/ml) ( Su et al., 2013 ); rabbit Skp2 antibody 4358 (Cell Signaling Technology, 1:50-200); mouse FLAG-M2 antibody F3165 (Sigma-Aldrich, 1:200-2000); rabbit HA antibody ab9110 (1:500-2000) and mouse β-actin ab6276 (1:5000) from Abcam; mouse monoclonal HA-tag 12ca5 antibody from the University of North Carolina Antibody Core Facility (1-2 μg/ml); rabbit Skp2 antibody sc-7164 (1:200), rabbit cyclin A sc-751 antibody (1:200), mouse E2F1 antibody sc-193 (1:200), rabbit p107 antibody sc-318 and rabbit p130 antibody sc-317 (1:200) from Santa Cruz; rabbit AR32 (1 μg/ml) ( Quarmby et al., 1990 ) and AR52 antipeptide antibodies (10 μg/ml) ( Lubahn et al., 1988 ).

Techniques: Sequencing, Binding Assay, Phospho-proteomics, Immunoprecipitation, Expressing, Transfection, Incubation, Western Blot

Journal: Molecular and cellular endocrinology

Article Title: Melanoma antigen-A11 regulates substrate-specificity of Skp2-mediated protein degradation

doi: 10.1016/j.mce.2016.10.006

Figure Lengend Snippet: [A] Inhibition of the MAGE-A11-cyclin A interaction by Skp2 was shown by immunoprecipitation by expressing 6 μg pCMV-FLAG or pCMV-FLAG-cyclin A/10 cm COS1 cell dish with or without 3 μg pSG5-MAGE and/or 4 μg pcDNA3-myc-Skp2. The day after transfection cells were incubated for 18 h in serum-free medium containing 10 ng/ml EGF. Immunoblots of cell extracts (60 μg protein/ml) and immunoprecipitates were probed using MAGE2, Skp2 and FLAG-M2 antibodies. [B] Inhibition of the Skp2-cyclin A interaction by p27Kip1 (p27) was shown by expressing 3 μg pCMV-FLAG (-) or pCMV-FLAG-Skp2 with or without 2 μg pcDNA3-HA-cyclin A alone or together with 3 μg pcDNA3-myc-p27 in COS1 cells. The day after transfection cells were incubated in serum-free medium containing 0.1 μg/ml EGF. Immunoblots of cell extracts (50 μg protein/lane) and immunoprecipitates were probed using HA ab9110, p27 and Skp2 antibodies. [C] Absence of inhibition of the MAGE-A11-cyclin A interaction by p27 was shown by expressing 5 μg pCMV-FLAG or pCMV-FLAG-cyclin A in 10 cm COS1 cell dishes with 3 μg pSG5-MAGE alone or together with 3 μg pcDNA-myc-p27. The day after transfection cells were placed in serum-free medium containing 10 ng/ml EGF. Cell extracts (60 μg protein/lane) and immunoprecipitates were probed on the immunoblots using MAGE2, FLAG-M2 and p27 antibodies.

Article Snippet: Antibodies used to probe the immunoblots included rabbit MAGE1 and MAGE2 against baculovirus expressed human FLAG-MAGE-A11 (0.5-10 μg/ml) ( Su et al., 2013 ); rabbit Skp2 antibody 4358 (Cell Signaling Technology, 1:50-200); mouse FLAG-M2 antibody F3165 (Sigma-Aldrich, 1:200-2000); rabbit HA antibody ab9110 (1:500-2000) and mouse β-actin ab6276 (1:5000) from Abcam; mouse monoclonal HA-tag 12ca5 antibody from the University of North Carolina Antibody Core Facility (1-2 μg/ml); rabbit Skp2 antibody sc-7164 (1:200), rabbit cyclin A sc-751 antibody (1:200), mouse E2F1 antibody sc-193 (1:200), rabbit p107 antibody sc-318 and rabbit p130 antibody sc-317 (1:200) from Santa Cruz; rabbit AR32 (1 μg/ml) ( Quarmby et al., 1990 ) and AR52 antipeptide antibodies (10 μg/ml) ( Lubahn et al., 1988 ).

Techniques: Inhibition, Immunoprecipitation, Expressing, Transfection, Incubation, Western Blot

Journal: Molecular and cellular endocrinology

Article Title: Melanoma antigen-A11 regulates substrate-specificity of Skp2-mediated protein degradation

doi: 10.1016/j.mce.2016.10.006

Figure Lengend Snippet: [A] Effects of MAGE-A11 on cyclin A were tested by expressing 3 μg pcDNA/10 cm dish of COS1 cells (lane 1) or 3 μg pcDNA-HA-cyclin A alone or with 0.25, 1 or 3 μg pSG5-MAGE with or without 4 μg pcDNA-myc-Skp2 as indicated. The day after transfection cells were incubated in serum-free medium containing 10 ng/ml EGF. Cell extracts (60 μg protein/lane) were probed on immunoblots using HA, Skp2, MAGE2 and β-actin antibodies. [B] Inhibition of Skp2-mediated E2F1 degradation by MAGE-A11 was shown by expressing 2 μg pCMV5 (-) or pCMV-E2F1 with 2 μg pSG5-MAGE and 2 μg pcDNA3-myc-hSkp2 alone or together in COS1 cells. After transfection cells were incubated overnight with 10 ng/ml EGF in serum-free medium. The immunoblot of cell extracts (40 μg protein/lane) was probed using E2F1, MAGE1 and Skp2 antibodies. [C] Increased degradation of retinoblastoma-related protein p130 but not p107 by Skp2 and MAGE-A11 was shown by expressing 2 μg CMV-p107 (lanes 1-4) or pcDNA-p130 (lanes 8-11) with or without 1 μg pcDNA-myc-Skp2 and/or 1 μg pSG5-MAGE in COS1 cells as indicated. The day after transfection cells were incubated in serum-free medium containing 0.1 μg/ml EGF. Cell extracts (40 μg protein/lane) were probed on immunoblots using p107, p130, Skp2 sc-7164 and MAGE1 antibodies.

Article Snippet: Antibodies used to probe the immunoblots included rabbit MAGE1 and MAGE2 against baculovirus expressed human FLAG-MAGE-A11 (0.5-10 μg/ml) ( Su et al., 2013 ); rabbit Skp2 antibody 4358 (Cell Signaling Technology, 1:50-200); mouse FLAG-M2 antibody F3165 (Sigma-Aldrich, 1:200-2000); rabbit HA antibody ab9110 (1:500-2000) and mouse β-actin ab6276 (1:5000) from Abcam; mouse monoclonal HA-tag 12ca5 antibody from the University of North Carolina Antibody Core Facility (1-2 μg/ml); rabbit Skp2 antibody sc-7164 (1:200), rabbit cyclin A sc-751 antibody (1:200), mouse E2F1 antibody sc-193 (1:200), rabbit p107 antibody sc-318 and rabbit p130 antibody sc-317 (1:200) from Santa Cruz; rabbit AR32 (1 μg/ml) ( Quarmby et al., 1990 ) and AR52 antipeptide antibodies (10 μg/ml) ( Lubahn et al., 1988 ).

Techniques: Expressing, Transfection, Incubation, Western Blot, Inhibition

Journal: Molecular and cellular endocrinology

Article Title: Melanoma antigen-A11 regulates substrate-specificity of Skp2-mediated protein degradation

doi: 10.1016/j.mce.2016.10.006

Figure Lengend Snippet: [A] An E2F1-MAGE-A11-Skp2 complex was shown by expressing 2 μg pSG5-MAGE with or without 2 μg pCMV-E2F1 and 3 μg pCMV-FLAG or pCMV-FLAG-Skp2 in 10 cm COS1 cell dishes. The day after transfection cells were incubated with 10 ng/ml EGF in serum-free medium. Immunoblots of cell extracts (40 μg protein/lane) and immunoprecipitates were probed using E2F1, MAGE2 and FLAG-M2 antibodies. [B] An E2F1-MAGE-A11-Skp2 complex was also shown by expressing 2 μg pSG5-MAGE with or without 3 μg pcDNA-myc-hSkp2 and pCMV-FLAG or pCMV-FLAG-E2F1 in COS1 cells. The day after transfection cells were incubated with 10 ng/ml EGF in serum-free medium. Immunoblots of cell extracts (60 μg protein/lane) and immunoprecipitates were probed using Skp2, MAGE2 and FLAG-M2 antibodies. [C] Effect of MAGE-A11 on Skp2 self-ubiquitination was investigated by immunoprecipitation after expressing 4 μg pcDNA3 (-) or pcDNA3-myc-hSkp2 with 3 μg pSG5-MAGE or 3 μg pCMV-p107 alone or together with 6 μg pCMV-FLAG (-) or pCMV-FLAG-ubiquitin (Flag-Ub) in COS1 cells. The day after transfection cells were incubated with 0.1 μg/ml EGF in serum-free medium. Immunoblots of cell extracts (40 μg protein/ml) and immunoprecipitates were probed using p107, Skp2 and MAGE1 antibodies.

Article Snippet: Antibodies used to probe the immunoblots included rabbit MAGE1 and MAGE2 against baculovirus expressed human FLAG-MAGE-A11 (0.5-10 μg/ml) ( Su et al., 2013 ); rabbit Skp2 antibody 4358 (Cell Signaling Technology, 1:50-200); mouse FLAG-M2 antibody F3165 (Sigma-Aldrich, 1:200-2000); rabbit HA antibody ab9110 (1:500-2000) and mouse β-actin ab6276 (1:5000) from Abcam; mouse monoclonal HA-tag 12ca5 antibody from the University of North Carolina Antibody Core Facility (1-2 μg/ml); rabbit Skp2 antibody sc-7164 (1:200), rabbit cyclin A sc-751 antibody (1:200), mouse E2F1 antibody sc-193 (1:200), rabbit p107 antibody sc-318 and rabbit p130 antibody sc-317 (1:200) from Santa Cruz; rabbit AR32 (1 μg/ml) ( Quarmby et al., 1990 ) and AR52 antipeptide antibodies (10 μg/ml) ( Lubahn et al., 1988 ).

Techniques: Expressing, Transfection, Incubation, Western Blot, Ubiquitin Proteomics, Immunoprecipitation

Journal: Molecular and cellular endocrinology

Article Title: Melanoma antigen-A11 regulates substrate-specificity of Skp2-mediated protein degradation

doi: 10.1016/j.mce.2016.10.006

Figure Lengend Snippet: Skp2-mediated protein degradation involves a cyclin A-cdk2-p27Kip1 complex that is regulated by MAGE-A11 interaction with Skp2, cyclin A and E2F1. MAGE-A11 contains a cyclin A peptide binding motif homologous to the cyclin A binding site in Skp2. MAGE-A11 interaction with cyclin A stabilizes cyclin A at low Skp2 levels, and promotes cyclin A degradation at higher levels of Skp2. MAGE-A11 interaction with cyclin A is not inhibited by the p27Kip1 cyclin-dependent kinase inhibitor that inhibits Skp2 interaction with cyclin A. However, MAGE-A11 interaction with cyclin A is inhibited by Skp2. MAGE-A11 mediates a stable complex between Skp2 and E2F1 that sequesters Skp2 and blocks Skp2-mediated down-regulation of E2F1.

Article Snippet: Antibodies used to probe the immunoblots included rabbit MAGE1 and MAGE2 against baculovirus expressed human FLAG-MAGE-A11 (0.5-10 μg/ml) ( Su et al., 2013 ); rabbit Skp2 antibody 4358 (Cell Signaling Technology, 1:50-200); mouse FLAG-M2 antibody F3165 (Sigma-Aldrich, 1:200-2000); rabbit HA antibody ab9110 (1:500-2000) and mouse β-actin ab6276 (1:5000) from Abcam; mouse monoclonal HA-tag 12ca5 antibody from the University of North Carolina Antibody Core Facility (1-2 μg/ml); rabbit Skp2 antibody sc-7164 (1:200), rabbit cyclin A sc-751 antibody (1:200), mouse E2F1 antibody sc-193 (1:200), rabbit p107 antibody sc-318 and rabbit p130 antibody sc-317 (1:200) from Santa Cruz; rabbit AR32 (1 μg/ml) ( Quarmby et al., 1990 ) and AR52 antipeptide antibodies (10 μg/ml) ( Lubahn et al., 1988 ).

Techniques: Binding Assay

Journal: Journal of Biological Chemistry

Article Title: A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

doi: 10.1074/jbc.m109.034223

Figure Lengend Snippet: FIGURE 1. RNA interference-mediated silencing of SKP1A in SN4741 cell line. A, SN4741 cells were infected for 1 week with LVs plasmid vectors encoding shRNAs targeting SKP1A, shRNA-1, and shRNA-2 or a scrambled sequence.RNAwasextractedandconvertedtosingle-strandedforanalyzingbyQ-PCR.Therelativeexpression level was assessed by normalizing to the housekeeping genes 18S-rRNA and -actin. The values are the means S.E. from two independent experiments conducted in two to five replicates. *, p 0.01 versus control (scrambled). B, after lentiviral infection cells homogenates were analyzed by Western blotting using Skp1 specific antibody. The bands were quantified by densitometry and normalized to -actin. C and D, scram- bled or shRNA-1 infected cells were grown with 10% FCS at 33 °C, and after fixation and permeabilization, Skp1 protein was detected by fluorescence microscopy. The images are representative fields from two independent experiments. The chart represents the mean immunoreactive densities of six to nine sepa- rate fields from two independent experiments normalized to number of cells in each field. *, p 0.01 versus control (scrambled).

Article Snippet: The cells were blocked with PBS containing 10% donkey serum at 37 °C for 1 h and incubated at 4 °C overnight with mouse monoclonal antibody against Skp1 p19 (H-6, antibody sc-5281, 1:25; Santa Cruz, CA), rabbit polyclonal antibody against ubiquitin (antibody ab19247, 1:100; Abcam, Cambridge, MA), mouse monoclonal anti-Hsc-70 antibody (antibody SM5072, 1:25; Acris Antibodies, Herford, Germany), mouse monoclonal -synuclein ( -synuclein) antibody (antibody 610786, 1:25; BDBiosciences Transduction Laboratories, Heidelberg, Germany), rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody (antibody AB152, 1:150; Millipore, Billerica, MA), mouse monoclonal anti- -tubulin antibody (antibody T6557, 1:10,000; Sigma), rabbit polyclonal antibody to 19 S regulator ATPase subunit Rpt3 (antibody PSMC4/S6b/ TBP7; PW8175, 1:150; Enzo Life Sciences, formerly Biomol, Plymouth, PA), all diluted in PBS containing 1% donkey serum and 0.05% Triton X-100.

Techniques: Infection, Plasmid Preparation, shRNA, Sequencing, Control, Western Blot, Fluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

doi: 10.1074/jbc.m109.034223

Figure Lengend Snippet: FIGURE2.EffectofSKP1Asilencingoncellcycleprogression.A,lentivirus-infectedSN4741cellswereseeded inDulbecco’smodifiedEagle’smediumwith10%FCSandpuromycinforselection.SKP1A-infectedcellsappear less dense than their corresponding control (scrambled vector). Pictures were acquired after 24 h, using an inverted microscope connected to a digital camera (20 objective). B, shRNA-1, shRNA-2, and scrambled vector infected cells were gently suspended in a hypotonic fluorochrome solution, incubated in the dark at 37 °C for 30 min, and analyzed for DNA content on a logarithmic scale by FACSCalibur flow cytometer with Cell Quest research software; 3 104 events/sample were acquired. Both shRNAs decreased the number of cells in G0/G1 and increased the number of cells in S phase, with a delay in completion the cell cycle. The values are the means S.E. from three independent experiments conducted in three replicates. *, p 0.05; **, p 0.01 versus control scrambled vector infected cells.

Article Snippet: The cells were blocked with PBS containing 10% donkey serum at 37 °C for 1 h and incubated at 4 °C overnight with mouse monoclonal antibody against Skp1 p19 (H-6, antibody sc-5281, 1:25; Santa Cruz, CA), rabbit polyclonal antibody against ubiquitin (antibody ab19247, 1:100; Abcam, Cambridge, MA), mouse monoclonal anti-Hsc-70 antibody (antibody SM5072, 1:25; Acris Antibodies, Herford, Germany), mouse monoclonal -synuclein ( -synuclein) antibody (antibody 610786, 1:25; BDBiosciences Transduction Laboratories, Heidelberg, Germany), rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody (antibody AB152, 1:150; Millipore, Billerica, MA), mouse monoclonal anti- -tubulin antibody (antibody T6557, 1:10,000; Sigma), rabbit polyclonal antibody to 19 S regulator ATPase subunit Rpt3 (antibody PSMC4/S6b/ TBP7; PW8175, 1:150; Enzo Life Sciences, formerly Biomol, Plymouth, PA), all diluted in PBS containing 1% donkey serum and 0.05% Triton X-100.

Techniques: Control, Plasmid Preparation, Inverted Microscopy, shRNA, Infection, Incubation, Flow Cytometry, Software

Journal: Journal of Biological Chemistry

Article Title: A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

doi: 10.1074/jbc.m109.034223

Figure Lengend Snippet: FIGURE 3. Effect of RNA interference-mediated SKP1A inhibition on the expression of dopaminergic markers. Following infection with SKP1A shRNA-1 or scrambled vector, the mRNA levels of ALDH1A1, HSPA8 (encoding Hsc-70), TH, DAT, and VMAT2 were quantified by Q-PCR. The relative expres- sion was assessed by normalizing to the housekeeping gene 18S-rRNA. The valuesarethemeansS.E.fromtwoindependentexperimentsconductedin two to five replicates. *, p 0.05; **, p 0.01 versus scrambled vector.

Article Snippet: The cells were blocked with PBS containing 10% donkey serum at 37 °C for 1 h and incubated at 4 °C overnight with mouse monoclonal antibody against Skp1 p19 (H-6, antibody sc-5281, 1:25; Santa Cruz, CA), rabbit polyclonal antibody against ubiquitin (antibody ab19247, 1:100; Abcam, Cambridge, MA), mouse monoclonal anti-Hsc-70 antibody (antibody SM5072, 1:25; Acris Antibodies, Herford, Germany), mouse monoclonal -synuclein ( -synuclein) antibody (antibody 610786, 1:25; BDBiosciences Transduction Laboratories, Heidelberg, Germany), rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody (antibody AB152, 1:150; Millipore, Billerica, MA), mouse monoclonal anti- -tubulin antibody (antibody T6557, 1:10,000; Sigma), rabbit polyclonal antibody to 19 S regulator ATPase subunit Rpt3 (antibody PSMC4/S6b/ TBP7; PW8175, 1:150; Enzo Life Sciences, formerly Biomol, Plymouth, PA), all diluted in PBS containing 1% donkey serum and 0.05% Triton X-100.

Techniques: Inhibition, Expressing, Infection, shRNA, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

doi: 10.1074/jbc.m109.034223

Figure Lengend Snippet: FIGURE 4. SKP1A overexpression decreases the susceptibility to MPP and proteasomal inhibition injury. SN4741 cells were stably transfected with expression vector pcDNA3.1hygro-SKP1A or empty expression vector, pcDNA(selectionwithhygromycin).A,representativegelmicrographofSkp1 protein levels evaluated by Western blot analysis. The autoradiogram-de- rived bands were quantified by densitometry, and the values of Skp1 were normalized to -actin and expressed as relative expression of control (pcDNA3.1hygro vector). B and C, transfected cells were injured with MPP (B, 150 or 250 M) for 48 h or with the proteasome inhibitor MG-132 (C, 12.5 and 25 M, dissolved in Me2SO) for 6 h. Cell viability was assessed by the MTT test. The results are the means S.E. of three to five independent experi- ments and expressed as percentages of control (pcDNA3.1hygro empty vec- tor). *, p 0.01 versus empty vector; #, p 0.01 versus respective control (without MPP or MG-132).

Article Snippet: The cells were blocked with PBS containing 10% donkey serum at 37 °C for 1 h and incubated at 4 °C overnight with mouse monoclonal antibody against Skp1 p19 (H-6, antibody sc-5281, 1:25; Santa Cruz, CA), rabbit polyclonal antibody against ubiquitin (antibody ab19247, 1:100; Abcam, Cambridge, MA), mouse monoclonal anti-Hsc-70 antibody (antibody SM5072, 1:25; Acris Antibodies, Herford, Germany), mouse monoclonal -synuclein ( -synuclein) antibody (antibody 610786, 1:25; BDBiosciences Transduction Laboratories, Heidelberg, Germany), rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody (antibody AB152, 1:150; Millipore, Billerica, MA), mouse monoclonal anti- -tubulin antibody (antibody T6557, 1:10,000; Sigma), rabbit polyclonal antibody to 19 S regulator ATPase subunit Rpt3 (antibody PSMC4/S6b/ TBP7; PW8175, 1:150; Enzo Life Sciences, formerly Biomol, Plymouth, PA), all diluted in PBS containing 1% donkey serum and 0.05% Triton X-100.

Techniques: Over Expression, Inhibition, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

doi: 10.1074/jbc.m109.034223

Figure Lengend Snippet: FIGURE 5. Differentiative features of naïve SN4741 cells. A, SN4741 cells carrying the temperature-sensitive mutant from the oncogene, SV40Tag-tsA58, have a fibroblast-like flat morphology at 33 °C. Under differentia- tion conditions (i.e. nonpermissive temperature of 39 °C and 0.5% FCS), the SN4741 cell line ceased prolifera- tion and after 48 h started to display a neuronal morphology with extensive neurite outgrowth. B, represent- ativehistogramsandpercentageofcellsatdifferentphasesofthecellcycleasanalyzedbyFACS.Thevaluesare the means S.E. from three independent experiments conducted in three replicates. *, p 0.01 versus control (33 °C, FCS 10%). C, SKP1A and DA neuron-specific markers were analyzed by Q-PCR in naïve SN4741. RNA was extracted from nondifferentiated and differentiated cells, and the expression levels of various DA neuron genes were assessed. The relative expression level was assessed by normalizing to -actin. The values are the means S.E. from two independent experiments conducted in two to five replicates. *, p 0.01 versus SN4741 cells treated under permissive conditions (10% FCS and 33 °C). D, after cell fixation and permeabilization, Skp1 protein was detected by fluorescence microscopy using a specific Skp1 primary antibody. The chart represents mean immunoreactive density of six to nine separate fields from two independent experiments normalized to number of cells in each field. *, p 0.05; **, p 0.01 versus SN4741 cells under permissive conditions.

Article Snippet: The cells were blocked with PBS containing 10% donkey serum at 37 °C for 1 h and incubated at 4 °C overnight with mouse monoclonal antibody against Skp1 p19 (H-6, antibody sc-5281, 1:25; Santa Cruz, CA), rabbit polyclonal antibody against ubiquitin (antibody ab19247, 1:100; Abcam, Cambridge, MA), mouse monoclonal anti-Hsc-70 antibody (antibody SM5072, 1:25; Acris Antibodies, Herford, Germany), mouse monoclonal -synuclein ( -synuclein) antibody (antibody 610786, 1:25; BDBiosciences Transduction Laboratories, Heidelberg, Germany), rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody (antibody AB152, 1:150; Millipore, Billerica, MA), mouse monoclonal anti- -tubulin antibody (antibody T6557, 1:10,000; Sigma), rabbit polyclonal antibody to 19 S regulator ATPase subunit Rpt3 (antibody PSMC4/S6b/ TBP7; PW8175, 1:150; Enzo Life Sciences, formerly Biomol, Plymouth, PA), all diluted in PBS containing 1% donkey serum and 0.05% Triton X-100.

Techniques: Mutagenesis, Control, Expressing, Fluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

doi: 10.1074/jbc.m109.034223

Figure Lengend Snippet: FIGURE 6. Differentiation-induced lethality of SKP1A-silenced cells. A, SKP1A silenced (shRNA-1 infected) and nonsilenced cells (scrambled) seeded in 10% FCS-containing medium were induced to differentiate for up to 48 h. There is a robust lethality in cultures of cells deficient in SKP1A that were induced to differentiate at restrictive temperature, compared with the viability and differentiative phenotype of scrambled vector-infected cells. The pictures were acquired using an inverted microscope connected to a digital camera (10 objective). B, cells were analyzed for DNA content by FACS. The percentage of cells in the G0/G1, S, and G2/M fractions was calcu- lated. The values are the means S.E. from three independent experiments conducted in three replicates. *, p 0.01 versus scrambled. C, differentiation with retinoic acid (10 M).

Article Snippet: The cells were blocked with PBS containing 10% donkey serum at 37 °C for 1 h and incubated at 4 °C overnight with mouse monoclonal antibody against Skp1 p19 (H-6, antibody sc-5281, 1:25; Santa Cruz, CA), rabbit polyclonal antibody against ubiquitin (antibody ab19247, 1:100; Abcam, Cambridge, MA), mouse monoclonal anti-Hsc-70 antibody (antibody SM5072, 1:25; Acris Antibodies, Herford, Germany), mouse monoclonal -synuclein ( -synuclein) antibody (antibody 610786, 1:25; BDBiosciences Transduction Laboratories, Heidelberg, Germany), rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody (antibody AB152, 1:150; Millipore, Billerica, MA), mouse monoclonal anti- -tubulin antibody (antibody T6557, 1:10,000; Sigma), rabbit polyclonal antibody to 19 S regulator ATPase subunit Rpt3 (antibody PSMC4/S6b/ TBP7; PW8175, 1:150; Enzo Life Sciences, formerly Biomol, Plymouth, PA), all diluted in PBS containing 1% donkey serum and 0.05% Triton X-100.

Techniques: shRNA, Infection, Plasmid Preparation, Inverted Microscopy

Journal: Journal of Biological Chemistry

Article Title: A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

doi: 10.1074/jbc.m109.034223

Figure Lengend Snippet: FIGURE 7. SKP1A-silencing causes inclusion body formation in differentiating cells. Scrambled and lentivirus-stable infected SN4741 cells grown on coverslips were induced to differentiate under restrictive conditions for 24 h (pre-lethal stage), fixed, and subjected to double immunostaining with anti--synuclein (red) and anti-TH(green)antibodies(AandB),anti--synuclein(red)andanti-ubiquitin(green)antibodies(CandD),anti--synuclein(red)andanti-PSMC4(green)antibodies(E), anti--tubulin (red) and anti-ubiquitin (green) antibodies (F), and anti--tubulin (red) and anti-PSMC4 (green) antibodies (G). Scrambled infected (control) cells show a diffusestainingpatternanddonotshowanyinclusionsatbothpermissiveandrestrictivetemperatures(A–D,upperimages).SKP1A-deficientcellsdevelopedmultiple and perinuclear (B, D, and E, bottom images, see arrowsand insets) inclusion bodies 24 h after induction of differentiation, with characteristics of aggresomes, staining for-tubulin(FandG).Thereactivityoftheaggregatestothedifferentantibodiesdemonstratedasimilarpatternasindicatedbyco-localizedyellowimmunostaining in the overlaid right-hand panel, with the addition of Topro staining (blue) to identify the nucleus. No fluorescence was detected when the primary antibody was omitted. Each panel shows a representative picture of 10–15 views in two independent experiments.

Article Snippet: The cells were blocked with PBS containing 10% donkey serum at 37 °C for 1 h and incubated at 4 °C overnight with mouse monoclonal antibody against Skp1 p19 (H-6, antibody sc-5281, 1:25; Santa Cruz, CA), rabbit polyclonal antibody against ubiquitin (antibody ab19247, 1:100; Abcam, Cambridge, MA), mouse monoclonal anti-Hsc-70 antibody (antibody SM5072, 1:25; Acris Antibodies, Herford, Germany), mouse monoclonal -synuclein ( -synuclein) antibody (antibody 610786, 1:25; BDBiosciences Transduction Laboratories, Heidelberg, Germany), rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody (antibody AB152, 1:150; Millipore, Billerica, MA), mouse monoclonal anti- -tubulin antibody (antibody T6557, 1:10,000; Sigma), rabbit polyclonal antibody to 19 S regulator ATPase subunit Rpt3 (antibody PSMC4/S6b/ TBP7; PW8175, 1:150; Enzo Life Sciences, formerly Biomol, Plymouth, PA), all diluted in PBS containing 1% donkey serum and 0.05% Triton X-100.

Techniques: Infection, Double Immunostaining, Ubiquitin Proteomics, Control, Staining, Fluorescence

Journal: Journal of Biological Chemistry

Article Title: A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

doi: 10.1074/jbc.m109.034223

Figure Lengend Snippet: FIGURE 8. Expression of Skp1 protein in MPTP-treated mouse midbrain and its abundance in brain tissue. C57/Bl mice (n 6–9) were treated with the parkinsonism-inducing neurotoxin MPTP (20 mg/kg/day) for 4 days followed by an additional 4-day resting period. A, mice midbrains homogenates were resolved by SDS-PAGE (4–15%gradient)andimmunoblottedwithspecificrabbitanti-Skp1andmouseanti-THantibodies.Representative blotimagesareshown.B,analysisofthebands,giveninarbitraryunits,isrepresentedgraphically.Thevaluesarethe meansS.E.fromtwoindependentexperiments.*,p0.01versusMPTP.C,therelativeabundanceofSkp1protein in mouse brain from naïve, untreated mice (n 5) was assessed by Western analysis in tissue lysates from different brain areas. FC, frontal cortex; Hip, hippocampus; St, striatum; Mb, midbrain.

Article Snippet: The cells were blocked with PBS containing 10% donkey serum at 37 °C for 1 h and incubated at 4 °C overnight with mouse monoclonal antibody against Skp1 p19 (H-6, antibody sc-5281, 1:25; Santa Cruz, CA), rabbit polyclonal antibody against ubiquitin (antibody ab19247, 1:100; Abcam, Cambridge, MA), mouse monoclonal anti-Hsc-70 antibody (antibody SM5072, 1:25; Acris Antibodies, Herford, Germany), mouse monoclonal -synuclein ( -synuclein) antibody (antibody 610786, 1:25; BDBiosciences Transduction Laboratories, Heidelberg, Germany), rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody (antibody AB152, 1:150; Millipore, Billerica, MA), mouse monoclonal anti- -tubulin antibody (antibody T6557, 1:10,000; Sigma), rabbit polyclonal antibody to 19 S regulator ATPase subunit Rpt3 (antibody PSMC4/S6b/ TBP7; PW8175, 1:150; Enzo Life Sciences, formerly Biomol, Plymouth, PA), all diluted in PBS containing 1% donkey serum and 0.05% Triton X-100.

Techniques: Expressing, SDS Page, Western Blot

Journal: Oncotarget

Article Title: SKP2 inactivation suppresses prostate tumorigenesis by mediating JARID1B ubiquitination

doi:

Figure Lengend Snippet: (A) Quantification analysis of average weights of anterior prostate (AP) tumors of mice at 5–6 months of age. The number of mice for each group is as indicated. The representative biopsies of AP from indicated genotypes of mice are shown in . (B) H&E staining of prostate tissues from indicated genotypes of mice at 5–6 months of age. Scale bars represent 100 μm. (C) Effects of Skp2 inactivation on the cell proliferation of Pten/Trp53 null MEFs. (D) Effects of Skp2 inactivation on soft agar transformation of Pten/Trp53 null MEFs. (E) Wound healing (migration) assay of Pten/Trp53 null MEFs upon SKP2 inactivation (Also see ). (F) Left panel, Western blot analysis of protein levels of JARID1B and H3K4me3 upon Skp2 inactivation in Pten/Trp53 null MEFs. Right panel, quantification analysis of protein levels for JARID1B and H3K4me3 in MEFs upon Skp2 inactivation. Error bars for C-F represent means ±SD.

Article Snippet: Tissue sections were probed with primary antibodies: mouse anti-SKP2 (1:250, Invitrogen, 2C8D9), rabbit anti-H3K4me3 (1:200, Cell Signaling, 9727).

Techniques: Staining, Transformation Assay, Migration, Western Blot

Journal: Oncotarget

Article Title: SKP2 inactivation suppresses prostate tumorigenesis by mediating JARID1B ubiquitination

doi:

Figure Lengend Snippet: (A) H&E and immunohistochemical (IHC) staining for H3K4me3 and JARID1B in prostate tumors of Pten/Trp53 and Pten/Trp53/Skp2 mutant mice. Scale bars represent 50 μm. (B) Quantification analysis of tumor cells positive for H3K4me3 and JARID1B. Error bars represent means ± SD from 3 mice for each group. (C) Modeling the recurrent growth of prostate cancer in Luc/Pten/Trp53 null mice with bioluminescence imaging (BLI). The changes of BLI signals of representative Luc/Pten/Trp53 mutant mouse were monitored prior to and post castration. Mouse castration was performed at 14 weeks of age. The regressive and recurrent tumor growth were monitored weekly and defined with the change of bioluminescence intensity in modeled mice (see ). (D) Quantification of BLI signaling changes in prostate tumors of castrated Luc/Pten/Trp53 mutant mice ( n = 3). The signal intensity was measured for regions of interest around the lower abdomen. (E) IHC staining of Skp2, H3K4me3 and Ki67 in regressive and recurrent lesions of prostate tumor of Luc/Pten/Trp53 mutant mice after castration. The regressive and recurrent tumor tissues of mice were collected at about 2 weeks or 3 weeks after castration, under the guidance of BLI. Scale bars represent 50 μm. (F) Quantification analysis of tumor cells positive for Skp2, H3K4me3 and Ki67. Error bars represent means ± SD from 3 mice for each group.

Article Snippet: Tissue sections were probed with primary antibodies: mouse anti-SKP2 (1:250, Invitrogen, 2C8D9), rabbit anti-H3K4me3 (1:200, Cell Signaling, 9727).

Techniques: Immunohistochemical staining, Immunohistochemistry, Mutagenesis, Imaging

Journal: Oncotarget

Article Title: SKP2 inactivation suppresses prostate tumorigenesis by mediating JARID1B ubiquitination

doi:

Figure Lengend Snippet: (A) Western blot analysis shows JARID1B elevation and H3K4me3 reduction in PC3 cells upon SKP2 knockdown by shRNA. S - short exposure, L - long exposure. Right panel: quantification analysis on the abundance of H3K4me3 and JARID1B proteins from the left panel. Error bars represent means ± SD. (B) Immunofluorescence images show the co-localization of endogenous SKP2 and JARID1B proteins in PC3 cells. Scale bars represent 10 μm. (C) Co-immunoprecipitation analysis shows a physical interaction between JARID1B and SKP2 proteins in HEK293T cells. (D) Co-immunoprecipitation analysis shows a physical interaction between endogenous JARID1B and SKP2 proteins in PC3 cells. (E) In vivo ubiquitination assay shows that a reduction of K63-linked ubiquitination of JARID1B upon addition of SKP2 in HEK293T cells. Cells were transfected with Flag-JARID1B, Myc-SKP2, along with various HA-ubiquitin (HA-Ub) constructs. K48 and K63 indicate HA-Ub-K48-only and HA-Ub-K63-only, respectively. WCL indicates whole cell lysates. (F) JARID1B mutation affects H3K4me3 levels by altering the ubiquitination. In vivo ubiquitination assay was performed in HEK293T cells transfected with HA-Ub-K63-only, along with various JARID1B constructs (Flag-JARID1B, Flag-JARID1B-K242R and Flag-JARID1B-K285R) (see ). Bottom panel: Western blot analysis shows the effects of JARID1B WT and JARID1B mutants on the changes of endogenous protein levels of H3K4me3 through the K63-linked ubiquitination in cells. WCL indicates the whole cell lysates.

Article Snippet: Tissue sections were probed with primary antibodies: mouse anti-SKP2 (1:250, Invitrogen, 2C8D9), rabbit anti-H3K4me3 (1:200, Cell Signaling, 9727).

Techniques: Western Blot, shRNA, Immunofluorescence, Immunoprecipitation, In Vivo, Ubiquitin Assay, Transfection, Construct, Mutagenesis

Journal: Oncotarget

Article Title: SKP2 inactivation suppresses prostate tumorigenesis by mediating JARID1B ubiquitination

doi:

Figure Lengend Snippet: (A) Immunofluorescence images show a co-localization of endogenous JARID1B and TRAF6 in PC3 cells. Scale bars represent 10 μm. (B) and (C) Co-immunoprecipitation analysis shows that endogenous TRAF6 physically interacts with JARID1B in PC3 cells, as shown by reciprocal co-immunoprecipitation between the two proteins (Also see ). (D) In vivo ubiquitination assay shows that TRAF6 increases K63-linked ubiquitination of JARID1B and SKP2 inhibits TRAF6-mediated JARID1B ubiquitination. Cells were transfected with Flag-JARID1B, HA-Ub-K63-only, Myc-TRAF6 and Myc-SKP2 constructs as indicated. WCL indicates the whole cell lysates. (E) TRAF6 mediates JARID1B ubiquitination through lysine residue 242. HEK293T cells were transfected with Flag-JARID1B WT or Flag-JARID1B-K242R, HA-Ub-K63-only, Myc-TRAF6 and Myc-SKP2 plasmids as indicated. In vivo ubiquitination assay was performed in a standard procedure. WCL indicates the whole cell lysates.

Article Snippet: Tissue sections were probed with primary antibodies: mouse anti-SKP2 (1:250, Invitrogen, 2C8D9), rabbit anti-H3K4me3 (1:200, Cell Signaling, 9727).

Techniques: Immunofluorescence, Immunoprecipitation, In Vivo, Ubiquitin Assay, Transfection, Construct

Journal: Oncotarget

Article Title: SKP2 inactivation suppresses prostate tumorigenesis by mediating JARID1B ubiquitination

doi:

Figure Lengend Snippet: (A) Immunofluorescence (IF) images show a co-localization of endogenous JARID1B and Fibrillarin (FBL) in nucleoli of PC3 cells upon SKP2 knockdown (Also see ). FBL indicates a nucleolar marker. Right panel: quantification of PC3 cells showing an increase of JARID1B localization in nucleolus. Error bars represent means ± SD. (B) IF images show a co-localization of endogenous JARID1B and K63-Ub in nucleoli of PC3 cells upon SKP2 knockdown. (C) Western blotting assay shows an increase of β-galactosidase (β-Gal) in PC3 cells upon SKP2 knockdown. (D) IF images show JARID1B in nucleolus as indicated by arrows and β-Gal in cytoplasm in senescent cells upon SKP2 knockdown. (E) The co-localization of endogenous JARID1B and Fibrillarin (FBL) in nucleoli of prostate tissues in Pten pc−/− ;Trp53 pc−/− ;Skp2 −/− mutant mice (Also see ). Scale bars represent 10 μm for panel A, B, D and E. (F) The positive staining of β-galactosidase in prostate tissues of Pten pc−/− ;Trp53 pc−/− ;Skp2 −/− mice but not in that of Pten pc−/− ;Trp53 pc−/− mice. Scale bars represent 50 μm.

Article Snippet: Tissue sections were probed with primary antibodies: mouse anti-SKP2 (1:250, Invitrogen, 2C8D9), rabbit anti-H3K4me3 (1:200, Cell Signaling, 9727).

Techniques: Immunofluorescence, Marker, Western Blot, Mutagenesis, Staining

Journal: Oncotarget

Article Title: SKP2 inactivation suppresses prostate tumorigenesis by mediating JARID1B ubiquitination

doi:

Figure Lengend Snippet: (A) Immunohistochemical staining on SKP2 and H3K4me3 in human prostate array tissues. Scale bars represent 50 μm. (B) Statistical analysis of the prostate tissue microarray stained with SKP2 and H3K4me3 antibodies. The percentages of different H3K4me3 levels were calculated for each level of SKP2 protein in 35 cases of human PCa specimens. SKP2 and H3K4me3 levels were graded as 0, 1, 2 and 3 by intensity scores. The H3K4me3 grades are color-coded. Numbers in parenthesis represent sample sizes. The statistical significance was determined by Chi-Square test . (C) A working model for the role of SKP2 on epigenetic regulation of JARID1B and H3K4me3 in PCa. SKP2 suppresses the activity of JARID1B by reducing its K63-linked ubiquitination through TRAF6 under oncogenic stimulation, leading to an elevated H3K4me3 thus contributes to PCa. SKP2 deficiency increases JARID1B transportation to nucleolus of cells through an increase of its ubiquitination, resulting in an induction of cellular senescence to suppress PCa tumorigenesis.

Article Snippet: Tissue sections were probed with primary antibodies: mouse anti-SKP2 (1:250, Invitrogen, 2C8D9), rabbit anti-H3K4me3 (1:200, Cell Signaling, 9727).

Techniques: Immunohistochemical staining, Staining, Microarray, Activity Assay

Journal: Molecular Oncology

Article Title: Lysosome‐dependent FOXA1 ubiquitination contributes to luminal lineage of advanced prostate cancer

doi: 10.1002/1878-0261.13497

Figure Lengend Snippet: Enhanced SKP2:FOXA1 interaction correlates with advanced PCa in human specimens. (A, B) Immunofluorescence (IF) images demonstrate the colocalization of SKP2 (green) and FOXA1 (red) proteins in primary prostate tumors. White arrows represent SKP2 and FOXA1 protein colocalization. Scale bars are 10 μm. (C) Immunohistochemistry (IHC) staining for SKP2 and FOXA1 in tissue microarray (TMA) of primary prostate tumors ( n = 80). The representative images shown are H&E, SKP2, and FOXA1. Black arrows represent high SKP2 and low FOXA1 protein levels. Red arrows represent low SKP2 and high FOXA1 protein. Scale bars are 100 μm. (D) Intensity scores for SKP2 and FOXA1 staining were graded as 0, 1, 2, and 3 and the respective statistical significance was determined by Chi‐square test (Fig. ) and Pearson correlation coefficient. (E) SKP2‐to‐FOXA1 ratio (SKP2:FOXA1) for IHC staining for SKP2 and FOXA1 TMA of primary prostate tumors ( n = 45). (F, G) SKP2 mRNA expression is elevated in recurrent PCa (GSE25136; P = 0.0459) and neuroendocrine PCa (NEPC) ( P = 0.0224). CRPC refers to Castration‐resistant PCa. Previously published PCa gene expression datasets were retrieved from GEO database ( ncbi.nlm.nih.gov/gds /) and cbioportal ( cbioportal.org ), respectively. The corresponding normalized SKP2 levels were plotted. (H) Immunoblot analysis for several PCa cell lines. Quantification analysis of the relative protein levels for SKP2 and SYP is displayed to the right ( n = 4). Comparison between groups was performed using Student's t ‐test. Bars indicate SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then incubated with primary antibodies including mouse anti‐SKP2 (2 μg·mL −1 , Invitrogen, 32‐3300), rabbit anti‐FOXA1 (1 : 250, Abcam, Cambridge, UK, ab170933), rat anti‐LAMP2 (1 : 1000, Invitrogen, MA1‐165), and mouse anti‐Myc tag (5 μg·mL −1 , Abcam, ab32) as previously described [ ].

Techniques: Immunofluorescence, Immunohistochemistry, Microarray, Staining, Expressing, Western Blot, Comparison

Journal: Molecular Oncology

Article Title: Lysosome‐dependent FOXA1 ubiquitination contributes to luminal lineage of advanced prostate cancer

doi: 10.1002/1878-0261.13497

Figure Lengend Snippet: SKP2 regulates FOXA1 through K6 and K29‐linked ubiquitination. (A, B) Immunoblot analysis displays FOXA1 elevation in C4‐2B ( n = 3) and 22Rv1 ( n = 3) cells upon SKP2 knockdown (KD) via shRNA. Quantification analysis of the relative protein levels for SKP2 and FOXA1 is displayed on the right. (C) Immunofluorescence (IF) images demonstrate the colocalization of endogenous SKP2 and FOXA1 proteins in C4‐2B and 22Rv1 PCa cells. Scale bars are 20 μm. (D, E) Co‐immunoprecipitation analysis displays a physical interaction for SKP2 and FOXA1 proteins in HEK293T ( n = 3) cells using Myc‐tagged SKP2 or Flag‐tagged FOXA1. WCL indicates the whole cell lysates. (F, G) Immunoprecipitation analysis displays an interaction for endogenous SKP2 and FOXA1 proteins in C4‐2B ( n = 3) and 22Rv1 ( n = 3) PCa cells. (H) In vivo ubiquitination assay displays an increase in HA‐Ub‐linked ubiquitination for FOXA1 by Myc‐SKP2 in HEK293T ( n = 3) cells. (I) In vivo ubiquitination assay demonstrates a reduction in ubiquitination for K6R and K29R mutants ( n = 3). (J) HEK293T cells were co‐transfected with Flag‐tagged FOXA1, HA‐ubiquitin (wild type, WT), and WT‐Myc‐SKP2 or a ▵F SKP2 mutant before being treated with Cycloheximide (CHX; 100 μg·mL −1 ) protein synthesis inhibitor for the indicated time points (h, hours). EV refers to empty vector. Lower panel is the corresponding plot for FOXA1 protein intensity ( n = 3). (K) The percent and mean fluorescence intensity (MFI) of FOXA1 ubiquitination in HEK293T cells after overexpression of Myc‐SKP2, Flag‐FOXA1, and HA‐Ubiquitin WT or mutants. Colored points represent number of replicates per group. Comparisons between groups were analyzed using paired two‐tailed Student's t ‐test. Bars indicate SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then incubated with primary antibodies including mouse anti‐SKP2 (2 μg·mL −1 , Invitrogen, 32‐3300), rabbit anti‐FOXA1 (1 : 250, Abcam, Cambridge, UK, ab170933), rat anti‐LAMP2 (1 : 1000, Invitrogen, MA1‐165), and mouse anti‐Myc tag (5 μg·mL −1 , Abcam, ab32) as previously described [ ].

Techniques: Western Blot, shRNA, Immunofluorescence, Immunoprecipitation, In Vivo, Ubiquitin Assay, Transfection, Mutagenesis, Plasmid Preparation, Fluorescence, Over Expression, Two Tailed Test

Journal: Molecular Oncology

Article Title: Lysosome‐dependent FOXA1 ubiquitination contributes to luminal lineage of advanced prostate cancer

doi: 10.1002/1878-0261.13497

Figure Lengend Snippet: Skp2 Abrogation elevates Foxa1 levels in vivo in mouse models. (A) Immunoblot analysis shows the effects of Skp2 on Foxa1 protein levels in mouse embryonic fibroblasts (MEFs) with indicated genotypes. Quantification of protein levels is relative to beta‐Actin and displayed in the right panel ( n = 3). (B, C) Immunofluorescence (IF) images show colocalization among Skp2, Foxa1, and ubiquitin in murine prostate organoids ( n = 3). White arrows indicate colocalization for Skp2, Foxa1, and Ubiquitin. Scale bars are 25 μm. (D) Western blotting analysis of Skp2 and Foxa1 protein levels for anterior prostate (AP) tissues of mice with the indicated genotypes. Quantification analysis for the corresponding protein expression is shown in the right panel ( n = 3). (E) Immunohistochemistry (IHC) staining of Skp2 and Foxa1 in prostate tissues of mice with the indicated genotypes. The side panel displays a quantification analysis for IHC staining ( n = 3). Scale bars are 100 μm. (F) IF images show colocalization (white arrows) among Skp2, Foxa1, and Pcna (proliferating cell nuclear antigen) prostate tissues in Pten pc−/− ; Trp53 pc−/− mutant mice, while Skp2 loss in Pten pc−/− ; Trp53 pc−/− ; Skp2 −/− mutant mice has decreased Skp2, Foxa1, and Pcna colocalization ( n = 3). Scale bars are 25 μm. (G) IF images for luminal (CD24 + ) and basal (CD49f + ) lineage markers in Pten pc−/− ; Trp53 pc−/− and Pten pc−/− ; Trp53 pc−/− ; Skp2 −/− mutant mice ( n = 3). Scale bars are 25 μm. Comparison between groups was performed using Student's t ‐test. Bars indicate SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then incubated with primary antibodies including mouse anti‐SKP2 (2 μg·mL −1 , Invitrogen, 32‐3300), rabbit anti‐FOXA1 (1 : 250, Abcam, Cambridge, UK, ab170933), rat anti‐LAMP2 (1 : 1000, Invitrogen, MA1‐165), and mouse anti‐Myc tag (5 μg·mL −1 , Abcam, ab32) as previously described [ ].

Techniques: In Vivo, Western Blot, Immunofluorescence, Expressing, Immunohistochemistry, Mutagenesis, Comparison

Journal: Molecular Oncology

Article Title: Lysosome‐dependent FOXA1 ubiquitination contributes to luminal lineage of advanced prostate cancer

doi: 10.1002/1878-0261.13497

Figure Lengend Snippet: SKP2 alters FOXA1 ubiquitination levels in human prostate cancer cells in a dose‐dependent manner. (A) Endogenous ubiquitination of FOXA1 in C4‐2B proceeding SKP2 inhibition using SZL P1‐41 at different concentrations (μ m ). Samples were collected and subjected to in vivo ubiquitination and western blot analysis ( n = 3). (B) FOXA1 ubiquitination profile using flow cytometry for vehicle, SKP2 KD, and SKP2 inhibition using SZL P1‐41 for C4‐2B. Representative histogram and percent ubiquitination of FOXA1 are plotted. Colored points represent number of replicates per group. (C) C4‐2B xenograft mice received vehicle (DMSO) or SKP2 inhibitor SZL P1‐41 (30 mg·kg −1 ; three times per week, intraperitoneal, i.p. injection) for 30 days. Immunofluorescence (IF) staining displays increased colocalization (white arrows) amongst SKP2, FOXA1, and PCNA for C4‐2B xenograft vehicle tissues, while exposure to SZL P1‐41 treatment reduced SKP2 and PCNA levels ( n = 3). Scale bars are 25 μm. (D) C4‐2B xenograft tissue sections were subjected to Immunohistochemistry (IHC) staining with the indicated antibodies ( n = 5). Scale bars are 100 μm. (E) IF luminal (CD24 + ) and basal (CD49f + ) lineage staining in C4‐2B vehicle and SZL P1‐41‐treated mice ( n = 3). Scale bars are 25 μm. Comparison between groups was performed using Student's t ‐test. Bars indicate SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then incubated with primary antibodies including mouse anti‐SKP2 (2 μg·mL −1 , Invitrogen, 32‐3300), rabbit anti‐FOXA1 (1 : 250, Abcam, Cambridge, UK, ab170933), rat anti‐LAMP2 (1 : 1000, Invitrogen, MA1‐165), and mouse anti‐Myc tag (5 μg·mL −1 , Abcam, ab32) as previously described [ ].

Techniques: Inhibition, In Vivo, Western Blot, Flow Cytometry, Injection, Immunofluorescence, Staining, Immunohistochemistry, Comparison

Journal: Molecular Oncology

Article Title: Lysosome‐dependent FOXA1 ubiquitination contributes to luminal lineage of advanced prostate cancer

doi: 10.1002/1878-0261.13497

Figure Lengend Snippet: FOXA1 Regulation by SKP2 is a lysosomal‐dependent event. (A) The effects of Chloroquine on FOXA1 levels in C4‐2B cells. Cells were subjected to MG132 or chloroquine treatment at the indicated concentrations (μ m ). Lower panels display the quantification for FOXA1 protein levels ( n = 3). (B) The effects of bafilomycin A1 (BA1) treatment at the indicated concentrations (μ m ) on FOXA1 protein levels in C4‐2B and 22Rv1 cells. Quantification for FOXA1 levels is displayed below ( n = 3). (C) Immunoblot demonstrating the effects of lysosomal marker (LC3, LAMP1, and LAMP2) KD on FOXA1 protein levels in C4‐2B and 22Rv1 cells. Lower panels demonstrate quantified FOXA1 protein levels ( n = 3). (D) Immunofluorescence (IF) images display an increase in LAMP2 (lysosome‐associated membrane glycoprotein) levels in C4‐2B cells upon Myc‐SKP2 overexpression ( n = 3). Scale bars are 20 μm. (E) CellLight Lysosome‐GFP, BacMam 2.0, labeling of lysosomal activity in association with FOXA1 in C4‐2B ( n = 3) and 22Rv1 ( n = 3). White arrows indicate colocalization between the lysosome and FOXA1. Scale bars are 25 μm. (F) IF images show the impact of Skp2 restoration on Lamp2 in Pten/Trp53/Skp2 triple‐null mouse embryonic fibroblasts (MEFs) proceeding transient overexpression of Myc‐SKP2 ( n = 3). White arrows indicate colocalization amongst Lamp2, FOXA1, and Skp2. Scale bars are 25 μm. (G) qRT‐PCR analysis of Skp2 and the lysosomal genes Hexb and Mcoln1 in Pten/Trp53/Skp2 triple‐null MEFs proceeding the transient overexpression of Myc‐SKP2 or an empty vector (EV). Results represent relative expression values to the housekeeping gene beta‐Actin ( n = 3). (H) Western blotting analysis of Skp2, FOXA1, and Lamp2 levels for Pten/Trp53/Skp2 triple‐null MEFs proceeding overexpression of Myc‐SKP2 (0–2 μg). Quantification analysis for the corresponding relative protein levels is shown in the right panel ( n = 3). (I) A working model on the K6‐ and K29‐linked ubiquitination of FOXA1 by SKP2 in lysosomal degradation. Comparison between groups was performed using Student's t ‐test. Bars indicate SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then incubated with primary antibodies including mouse anti‐SKP2 (2 μg·mL −1 , Invitrogen, 32‐3300), rabbit anti‐FOXA1 (1 : 250, Abcam, Cambridge, UK, ab170933), rat anti‐LAMP2 (1 : 1000, Invitrogen, MA1‐165), and mouse anti‐Myc tag (5 μg·mL −1 , Abcam, ab32) as previously described [ ].

Techniques: Western Blot, Marker, Immunofluorescence, Membrane, Over Expression, Labeling, Activity Assay, Quantitative RT-PCR, Plasmid Preparation, Expressing, Comparison

Journal: iScience

Article Title: A LY6E-PHB1-TRIM21 assembly degrades CD14 protein to mitigate LPS-induced inflammatory response

doi: 10.1016/j.isci.2023.106808

Figure Lengend Snippet: TRIM21 is the primary E3 ligase recruited by PHB1 to participate in the LY6E-meidated ubiquitination of CD14 (A) PHB1-mediated connection between CD14 and a ubiquitin E3 ligase. HEK-293T cells stably expressing non-tagged LY6E or control cells described in Figure 2 C were infected with control shRNA or PHB1-specific shRNA (sh2, denoted as shPHB1) encoding lentiviral vector, prior to transfection with a plasmid expressing FLAG-CD14 or an empty plasmid. The cells were used for preparation of cell extracts that were subjected to selection by anti-FLAG affinity gel, and the resultant immunoprecipitaes were analyzed for the presence of the indicated proteins, including two known PHB1-interacting E3 ligases, SKP2 and TRIM21, by immunoblotting. (B) Determination of the role of TRIM21 in LY6E-mediated CD14 ubiquitination. The effect of the expression of TRIM21-specific shRNA (shTRIM21) on the ubiquitination of transfected FLAG-CD14 was evaluated in the HEK-293T cells stably expressing non-tagged LY6E, using the procedures detailed in Figure 2 D. (C) Examination of TRIM21 dependency of LY6E-mediated CD14 suppression in THP-1 cells. LY6E-overexpressing THP-1 cells were enforced to express control shRNA (shCtrl) or TRIM21-specific shRNA (shTRIM21) by lentiviral vector infection, and analyzed for CD14 surface expression by flow cytometry. Shown are representative flow cytometric plots ( top ) and aggregate data ( bottom ) on the percentage of CD14 positive cells and MFI (n = 3; ∗∗∗∗, p < 0.0001; one-way ANOVA). (D) Dissection of the relationship of LY6E and PHB1 in interacting with CD14. Wild-type HEK-293T were infected with an empty lentiviral vector or vectors encoding either a LY6E-targeting shRNA (shLY6E) or non-tagged LY6E, and subsequently mock transfected or transfected with an FLAG-CD14 expression plasmid. The cells were analyzed for the association between CD14 and LY6E, PHB1 by anti-FLAG co-immunoprecipitations followed by immunoblotting with the indicated antibodies. Cells under LY6E overexpression were also treated with MG132 to stabilize the co-expressed CD14 proteins.

Article Snippet: SKP2 Rabbit pAb , ABclonal , Cat# A7728; AB_2772237.

Techniques: Ubiquitin Proteomics, Stable Transfection, Expressing, Control, Infection, shRNA, Plasmid Preparation, Transfection, Selection, Western Blot, Flow Cytometry, Dissection, Over Expression

Journal: iScience

Article Title: A LY6E-PHB1-TRIM21 assembly degrades CD14 protein to mitigate LPS-induced inflammatory response

doi: 10.1016/j.isci.2023.106808

Figure Lengend Snippet:

Article Snippet: SKP2 Rabbit pAb , ABclonal , Cat# A7728; AB_2772237.

Techniques: Recombinant, Software, Membrane, Protein Extraction

Journal: iScience

Article Title: A nonautophagic role of ATG5 in regulating cell growth by targeting c-Myc for proteasome-mediated degradation

doi: 10.1016/j.isci.2021.103296

Figure Lengend Snippet:

Article Snippet: Antibodies against ATG5 (mouse), USP28, SKP2, TRIM32, CHIP, and the His tag were purchased from Proteintech (China).

Techniques: Virus, shRNA, Recombinant, Plasmid Preparation, Software, Luciferase, Reporter Gene Assay, Bicinchoninic Acid Protein Assay, Reverse Transcription