Journal: Cell death and differentiation

Article Title: CDK15 promotes colorectal cancer progression via phosphorylating PAK4 and regulating β-catenin/ MEK-ERK signaling pathway.

doi: 10.1038/s41418-021-00828-6

Figure Lengend Snippet: Fig. 5 PAK4 mediates the oncogenic effect of CDK15 in colorectal cancer. A p-β-catenin(Ser675), c-Myc, p-MEK1/2 (Ser217/221), and p-ERK1/ 2 (Thr202/Tyr204) were detected by western blot after CDK15 knockdown in CCD18-co, SW480 and HCT116 cells. B Cells with PAK4 silencing and CDK15 overexpression were established. PAK4 and CDK15 expression was determined by western blot. C PAK4 knockdown reverses cell proliferation induced by CDK15 in CCD18-co and HCT116 cells. MTT assay was used to detect cell proliferation. D Anchorage-independent growth in CCD18-co and HCT116 cells with PAK4 silencing and CDK15 overexpression. Left panels: representative images (Scale bar: 200 μm). Right panels: Colonies were counted using Image J-Plus (Scale bar: 200 μm) and data represented statistical analysis of colony number ratio. E Anchorage-independent growth in CCD18-co and HCT116 cells treated with PAK4 inhibitor (PF-3758309). Left panel: representative images of colonies (Scale bar: 200 μm). Right panel: statistical analysis of the colony ratio. F Western blot to validate β-catenin and MEK/ERK signaling pathway in HCT116 cells with indicated treatment. Data were presented as mean values ± SD from triplicate experiments. Statistical differences were evaluated using Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The following antibodies were used in our study: anti-CDK15 (Cat#PA5-28595, Invitrogen), anti-CDK15 (Cat#TA811934, ORIGENE), anti-PAK4 (Cat#sc-390507, Santa Cruz), anti- phospho-β-catenin (Ser675) (Cat#4176, Cell Signaling Technology), anti-β-catenin (Cat # 8480, Cell Signaling Technology), antiphospho-ERK1/2 (Thr202/Tyr204)(Cat# 4370, Cell Signaling Technology), Anti-ERK1/2 (Cat#4695, Cell Signaling Technology), anti-phospho-MEK1/2 (Ser217/221)(Cat# 9154, Cell Signaling Technology), anti-MEK1/2 (Cat# 4694, Cell Signaling Technology), anti-c-Myc antibody (Cat#ab32072, Abcam), anti-Flag (Cat #F1804, Sigma), anti-HA (Cat #3724, Cell Signaling Technology), anti-His (Cat #ab137839, Abcam), anti-GAPDH (#HRP-60004, Proteintech), anti-β-actin (#HRP-60008, Proteintech).

Techniques: Western Blot, Knockdown, Over Expression, Expressing, MTT Assay

Journal: Cell death and differentiation

Article Title: CDK15 promotes colorectal cancer progression via phosphorylating PAK4 and regulating β-catenin/ MEK-ERK signaling pathway.

doi: 10.1038/s41418-021-00828-6

Figure Lengend Snippet: Fig. 7 Targeting PAK4 delays tumor growth in patient-derived xenografts. A Clinical information for HJG208 and HJG210 from patient’s cancer tissues. B, C 0.9% NaCl as vehicle, 5 mg/kg or 20 mg/kg PF-3758309 were intraperitoneally injected once per day for 20 days, and tumor volume was monitored every 2–5 days (n = 9–10 mice per group). D, E Tumor photographs. F, G Tumor weight and tumor growth inhibition (H, I) normalized to control group. J Levels of p-β-catenin (Ser675), c-Myc, p-MEK1/2 (Ser217/221), and p-ERK1/2 (Thr202/Tyr204) in harvested tumor tissues were assessed by immunohistochemistry. Representative photographs for each antibody in different groups are shown (100×; Scale bar: 50 μm). K Statistical analysis for immunohistochemistry staining. Statistical differences were evaluated using Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent mean ± SD.

Article Snippet: The following antibodies were used in our study: anti-CDK15 (Cat#PA5-28595, Invitrogen), anti-CDK15 (Cat#TA811934, ORIGENE), anti-PAK4 (Cat#sc-390507, Santa Cruz), anti- phospho-β-catenin (Ser675) (Cat#4176, Cell Signaling Technology), anti-β-catenin (Cat # 8480, Cell Signaling Technology), antiphospho-ERK1/2 (Thr202/Tyr204)(Cat# 4370, Cell Signaling Technology), Anti-ERK1/2 (Cat#4695, Cell Signaling Technology), anti-phospho-MEK1/2 (Ser217/221)(Cat# 9154, Cell Signaling Technology), anti-MEK1/2 (Cat# 4694, Cell Signaling Technology), anti-c-Myc antibody (Cat#ab32072, Abcam), anti-Flag (Cat #F1804, Sigma), anti-HA (Cat #3724, Cell Signaling Technology), anti-His (Cat #ab137839, Abcam), anti-GAPDH (#HRP-60004, Proteintech), anti-β-actin (#HRP-60008, Proteintech).

Techniques: Derivative Assay, Injection, Inhibition, Control, Immunohistochemistry, Staining

Journal: Cell death and differentiation

Article Title: CDK15 promotes colorectal cancer progression via phosphorylating PAK4 and regulating β-catenin/ MEK-ERK signaling pathway.

doi: 10.1038/s41418-021-00828-6

Figure Lengend Snippet: Fig. 8 Lentivirus-mediated CDK15 silencing inhibits colorectal tumor growth in patient-derived xenografts. A Clinical information for HJG86 from patient’s cancer tissues. B Mice received the lentiviruses (shNT, shCDK15-3, shCDK15-7) via intratumoral injection every 3 days for a total of four times. Tumor volume was monitored every 2–5 days for four continuous weeks (n = 8 mice per group). C Tumors photographs. D Tumor weight measured at the end of the study. E Tumor growth inhibition normalized to control group. F Levels of CDK15, p-β-catenin (Ser675), c-Myc, p-MEK1/2(Ser217/221), and p-ERK1/2 (Thr202/Tyr204) in harvested tissues were assessed by immunohistochemistry. Representative photographs in different groups are shown (100×; Scale bar: 50 μm). G Statistical analysis for immunohistochemistry staining. H Schematic model for the findings of this work: Aberrant CDK15 in CRC binds PAK4 and phosphorylates PAK4 at the S291 site. Accumulated phosphorylation of S291 upregulates the β-catenin/c-Myc and MEK/ERK signals, which in turn contribute to CRC tumor growth. Statistical differences were evaluated using Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent mean ± SD.

Article Snippet: The following antibodies were used in our study: anti-CDK15 (Cat#PA5-28595, Invitrogen), anti-CDK15 (Cat#TA811934, ORIGENE), anti-PAK4 (Cat#sc-390507, Santa Cruz), anti- phospho-β-catenin (Ser675) (Cat#4176, Cell Signaling Technology), anti-β-catenin (Cat # 8480, Cell Signaling Technology), antiphospho-ERK1/2 (Thr202/Tyr204)(Cat# 4370, Cell Signaling Technology), Anti-ERK1/2 (Cat#4695, Cell Signaling Technology), anti-phospho-MEK1/2 (Ser217/221)(Cat# 9154, Cell Signaling Technology), anti-MEK1/2 (Cat# 4694, Cell Signaling Technology), anti-c-Myc antibody (Cat#ab32072, Abcam), anti-Flag (Cat #F1804, Sigma), anti-HA (Cat #3724, Cell Signaling Technology), anti-His (Cat #ab137839, Abcam), anti-GAPDH (#HRP-60004, Proteintech), anti-β-actin (#HRP-60008, Proteintech).

Techniques: Derivative Assay, Injection, Inhibition, Control, Immunohistochemistry, Staining, Phospho-proteomics

Journal: Molecular medicine reports

Article Title: Twist-related protein 1 enhances oral tongue squamous cell carcinoma cell invasion through β-catenin signaling.

doi: 10.3892/mmr.2014.2904

Figure Lengend Snippet: Figure 1. TWIST expression in OTSCC cells with overexpression and knockdown of TWIST. (A) SCC‑4 and (B) TCA8113 human OTSCC cells. Lane 1, NC; lane 2, VC; lane 3, T; lane 4, T+B‑shRNA; lane 5, SC; lane 6, T‑shRNA. Expression was analyzed with western blot analysis. β‑actin was used as a loading control. The density of the TWIST blots were normalized against that of β‑actin blots to obtain a relative blot density. This was expressed as a fold change of the relative TWIST blot density compared with the NC group (designated as 1). aP<0.05, compared with NC or VC, bP<0.05, compared with T, cP<0.05, compared with T+B‑shRNA and dP<0.05, compared with SC. OTSCC, oral tongue squamous cell carcinoma; NC, normal control; VC, cells transfected with empty pcDNA3 vector; T, cells transfected with pcDNA3‑TWIST expression vector; T+B‑shRNA, cells transfected with pcDNA‑TWIST expression vector and β‑catenin short hairpin RNA; SC, cells transfected with scrambled control shRNA; T‑shRNA, cells transfected with TWIST shRNA; TWIST, twist‑related protein 1.

Article Snippet: Twist (sc-38604-V) and β-catenin (sc-29209-V) short hairpin (sh) RNA lentiviral particles, control shRNA lentiviral particles-A (sc-108080), anti-TWIST (sc-81417) mouse monoclonal antibodies, anti-β-catenin (sc-7963) mouse monoclonal antibodies and anti-matrix metalloproteinase-2 (MMP-2, sc-53630) mouse monoclonal antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Over Expression, Knockdown, Western Blot, Control, Transfection, Plasmid Preparation, shRNA

Journal: Molecular medicine reports

Article Title: Twist-related protein 1 enhances oral tongue squamous cell carcinoma cell invasion through β-catenin signaling.

doi: 10.3892/mmr.2014.2904

Figure Lengend Snippet: Figure 2. Effect of TWIST on β‑catenin luciferase reporter activity in OTSCC cells. (A) SCC‑4 and (B) TCA8113 human OTSCC cells were transfected with TOPflash, a synthetic β‑catenin luciferase reporter, or FOPflash, a negative control reporter for TOPflash. After 24 h, the luciferase activity in each group was analyzed. The luciferase activity was expressed as a fold change relative to that of the NC group (designated as 1). aP<0.05, compared with NC or VC, bP<0.05, compared with T, cP<0.05, compared with B‑shRNA, dP<0.05, compared with T+B‑shRNA and eP<0.05, compared with SC. OTSCC, oral tongue squamous cell carcinoma; NC, normal control; VC, cells transfected with empty pcDNA3 vector; T, cells transfected with pcDNA3‑TWIST expression vector; T+B‑shRNA, cells transfected with pcDNAs‑TWIST expression vector and β‑catenin short hairpin RNA; SC, cells transfected with scrambled control shRNA; T‑shRNA, cells transfected with TWIST shRNA; TWIST, twist‑related protein 1.

Article Snippet: Twist (sc-38604-V) and β-catenin (sc-29209-V) short hairpin (sh) RNA lentiviral particles, control shRNA lentiviral particles-A (sc-108080), anti-TWIST (sc-81417) mouse monoclonal antibodies, anti-β-catenin (sc-7963) mouse monoclonal antibodies and anti-matrix metalloproteinase-2 (MMP-2, sc-53630) mouse monoclonal antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Luciferase, Activity Assay, Transfection, Negative Control, Control, Plasmid Preparation, Expressing, shRNA

Journal: Molecular medicine reports

Article Title: Twist-related protein 1 enhances oral tongue squamous cell carcinoma cell invasion through β-catenin signaling.

doi: 10.3892/mmr.2014.2904

Figure Lengend Snippet: Figure 3. Effect of TWIST on mRNA levels of β‑catenin, C‑Myc and C‑Jun in OTSCC cells. (A) SCC‑4 and (B) TCA8113 human OTSCC. Reverse transcrip tion‑qunatitative polymerase chain reaction was performed in each group. The mRNA levels were expressed as a fold change relative to that of the NC group (designated as 1). aP<0.05, compared with NC or VC. OTSCC, oral tongue squamous cell carcinoma; NC, normal control; VC, cells transfected with empty pcDNA3 vector; T, cells transfected with pcDNA3‑TWIST expression vector; T+B‑shRNA, cells transfected with pcDNAs‑TWIST expression vector and β‑catenin short hairpin RNA; T+Ly, cells transfected with pcDNAs‑WIST expression vector and treated with 50 µm LY294002; SC, cells transfected with scrambled control shRNA; T‑shRNA, cells transfected with TWIST shRNA; TWIST, twist‑related protein 1.

Article Snippet: Twist (sc-38604-V) and β-catenin (sc-29209-V) short hairpin (sh) RNA lentiviral particles, control shRNA lentiviral particles-A (sc-108080), anti-TWIST (sc-81417) mouse monoclonal antibodies, anti-β-catenin (sc-7963) mouse monoclonal antibodies and anti-matrix metalloproteinase-2 (MMP-2, sc-53630) mouse monoclonal antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Polymerase Chain Reaction, Control, Transfection, Plasmid Preparation, Expressing, shRNA

Journal: Molecular medicine reports

Article Title: Twist-related protein 1 enhances oral tongue squamous cell carcinoma cell invasion through β-catenin signaling.

doi: 10.3892/mmr.2014.2904

Figure Lengend Snippet: Figure 4. Effect of TWIST on levels of β‑catenin protein in OTSCC cells. (A) SCC‑4 and (B) TCA8113 human OTSCC cells. Lane 1, NC; lane 2, VC; lane 3, T; lane 4, B‑shRNA; lane 5, T+B‑shRNA; lane 6, T+LY; lane 7, SC; lane 8, T‑shRNA. The soluble and total β‑catenin protein levels were analyzed with western blot analysis. β‑actin was used as a loading control. The density of the β‑catenin blot was normalized against that of β‑actin to obtain a relative blot density, which was expressed as a fold change to the relative β‑catenin blot density in the NC group (designated as 1). aP<0.05, compared with NC or VC, bP<0.05, compared with T, cP<0.05, compared with B‑shRNA, dP<0.05, compared with T+B‑shRNA, eP<0.05, compared with T+LY and fP<0.05, compared with SC. OTSCC, oral tongue squamous cell carcinoma; NC, normal control; VC, cells transfected with empty pcDNA3 vector; T, cells transfected with pcDNA3‑TWIST expression vector; T+B‑shRNA, cells transfected with pcDNAs‑TWIST expression vector and β‑catenin short hairpin RNA; T+Ly, cells transfected with pcDNAs‑WIST expression vector and treated with 50 µm LY294002; SC, cells transfected with scrambled control shRNA; T‑shRNA, cells transfected with TWIST shRNA; TWIST, twist‑related protein 1.

Article Snippet: Twist (sc-38604-V) and β-catenin (sc-29209-V) short hairpin (sh) RNA lentiviral particles, control shRNA lentiviral particles-A (sc-108080), anti-TWIST (sc-81417) mouse monoclonal antibodies, anti-β-catenin (sc-7963) mouse monoclonal antibodies and anti-matrix metalloproteinase-2 (MMP-2, sc-53630) mouse monoclonal antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Western Blot, Control, Transfection, Plasmid Preparation, Expressing, shRNA

Journal: Molecular medicine reports

Article Title: Twist-related protein 1 enhances oral tongue squamous cell carcinoma cell invasion through β-catenin signaling.

doi: 10.3892/mmr.2014.2904

Figure Lengend Snippet: Figure 5. Effect of TWIST on phosphorylated GSK‑3β levels in OTSCC cells. (A) SCC‑4 and (B) TCA8113 human OTSCC cells. Lane 1, NC; lane 2, VC; lane 3, T; lane 4, B‑shRNA; lane 5, T+B‑shRNA; lane 6, T+LY; lane 7, SC; lane 8, T‑shRNA. Phosphorylation of GSK‑3β at serine 9 in each group was analyzed with western blotting. β‑actin was used as a loading control. The density of the P‑GSK‑3β blot was normalized against that of total GSK‑3β and β‑actin to obtain a relative blot density, which was expressed as a fold change of the relative P‑GSK‑3β blot density in the NC group (designated as 1). aP<0.05, compared with NC or VC, bP<0.05, compared with T, cP<0.05, compared with B‑shRNA, dP<0.05, compared with T+B‑shRNA, eP<0.05, compared with T+LY and fP<0.05, compared with SC. P‑GSK‑3β, phosphorylated glycogen synthase‑3β; OTSCC, oral tongue squamous cell carcinoma; NC, normal control; VC, cells transfected with empty pcDNA3 vector; T, cells transfected with pcDNA3‑TWIST expression vector; T+B‑shRNA, cells transfected with pcDNAs‑TWIST expression vector and β‑catenin short hairpin RNA; T+Ly, cells transfected with pcDNAs‑WIST expression vector and treated with 50 µm LY294002; SC, cells transfected with scrambled control shRNA; T‑shRNA, cells transfected with TWIST shRNA; TWIST, twist‑related protein 1.

Article Snippet: Twist (sc-38604-V) and β-catenin (sc-29209-V) short hairpin (sh) RNA lentiviral particles, control shRNA lentiviral particles-A (sc-108080), anti-TWIST (sc-81417) mouse monoclonal antibodies, anti-β-catenin (sc-7963) mouse monoclonal antibodies and anti-matrix metalloproteinase-2 (MMP-2, sc-53630) mouse monoclonal antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Phospho-proteomics, Western Blot, Control, Transfection, Plasmid Preparation, Expressing, shRNA

Journal: Molecular medicine reports

Article Title: Twist-related protein 1 enhances oral tongue squamous cell carcinoma cell invasion through β-catenin signaling.

doi: 10.3892/mmr.2014.2904

Figure Lengend Snippet: Figure 7. Effect of TWIST on expression of MMP‑2 in OTSCC cells. (A) SCC‑4 and (B) TCA8113 human OTSCC cells. Lane 1, NC; lane 2, VC; lane 3, T; lane 4, B‑shRNA; lane 5, T+B‑shRNA; lane 6, T+LY; lane 7, SC; lane 8, T‑shRNA. The expression of MMP‑2 in each group was measured using western blot analysis. β‑actin was used as a loading control. The density of the MMP‑2 blot was normalized against that of β‑actin to obtain a relative blot density, which was expressed as a fold change of the relative MMP‑2 blot density in the NC group (designated as 1). aP<0.05, compared with NC or VC, bP<0.05, compared with T, cP<0.05, compared with B‑shRNA, dP<0.05, compared with T+B‑shRNA, eP<0.05, compared with T+LY and fP<0.05, compared with SC. MMP‑2, matrix metalloproteinase‑2, OTSCC, oral tongue squamous cell carcinoma; NC, normal control; VC, cells transfected with empty pcDNA3 vector; T, cells transfected with pcDNA3‑TWIST expression vector; T+B‑shRNA, cells transfected with pcDNAs‑TWIST expression vector and β‑catenin short hairpin RNA; T+Ly, cells transfected with pcDNAs‑WIST expression vector and treated with 50 µm LY294002; SC, cells transfected with scrambled control shRNA; T‑shRNA, cells transfected with TWIST shRNA; TWIST, twist‑related protein 1.

Article Snippet: Twist (sc-38604-V) and β-catenin (sc-29209-V) short hairpin (sh) RNA lentiviral particles, control shRNA lentiviral particles-A (sc-108080), anti-TWIST (sc-81417) mouse monoclonal antibodies, anti-β-catenin (sc-7963) mouse monoclonal antibodies and anti-matrix metalloproteinase-2 (MMP-2, sc-53630) mouse monoclonal antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Control, Transfection, Plasmid Preparation, shRNA

Journal: Molecular medicine reports

Article Title: Twist-related protein 1 enhances oral tongue squamous cell carcinoma cell invasion through β-catenin signaling.

doi: 10.3892/mmr.2014.2904

Figure Lengend Snippet: Figure 6. Effect of TWIST on OTSCC cells. (A) SCC‑4 and (B) TCA8113 human OTSCC cells. Transwell invasion assays were performed in each group and the number of cells that had invaded were counted. aP<0.05, cpmpared with NC or VC, bP<0.05, compared with T, cP<0.05, compared with B‑shRNA, dP<0.05, compared with T+B‑shRNA, eP<0.05, compared with T+LY and fP<0.05, compared with SC. OTSCC, oral tongue squamous cell carcinoma; NC, normal control; VC, cells transfected with empty pcDNA3 vector; T, cells transfected with pcDNA3‑TWIST expression vector; T+B‑shRNA, cells transfected with pcDNAs‑TWIST expression vector and β‑catenin short hairpin RNA; T+Ly, cells transfected with pcDNAs‑WIST expression vector and treated with 50 µm LY294002; SC, cells transfected with scrambled control shRNA; T‑shRNA, cells transfected with TWIST shRNA; TWIST, twist‑related protein 1.

Article Snippet: Twist (sc-38604-V) and β-catenin (sc-29209-V) short hairpin (sh) RNA lentiviral particles, control shRNA lentiviral particles-A (sc-108080), anti-TWIST (sc-81417) mouse monoclonal antibodies, anti-β-catenin (sc-7963) mouse monoclonal antibodies and anti-matrix metalloproteinase-2 (MMP-2, sc-53630) mouse monoclonal antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Control, Transfection, Plasmid Preparation, Expressing, shRNA

Journal: The Journal of cell biology

Article Title: The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium.

doi: 10.1083/jcb.201408060

Figure Lengend Snippet: Figure 4. Rpgrip1l deficiency causes impaired proteasomal activity at primary cilia. (A–F and I–L) MEFs were isolated from E12.5 WT and Rpgrip1l−/− embryos. (A and B) Western blot analysis of WT and Rpgrip1l−/− MEF lysates (n = 4 embryos, respectively). (C) Western blot analysis of WT and Rpgrip1l−/− MEF lysates (n = 3 embryos, respectively). (A–C) Actin serves as a loading control. (A) Phospho-(S33/37/T41)-β-Catenin is significantly increased in serum-starved Rpgrip1l−/− MEF lysates (82% of all cells had cilia; in serum-starved Rpgrip1l+/+ MEF lysates, 88.67% of all cells possessed cilia) but not in non–serum-starved Rpgrip1l−/− MEF lysates (4% of all cells displayed cilia; in non–serum-starved Rpgrip1l+/+ MEF lysates, 6.67% of all cells carried cilia; C). (B) Non–phospho-(S33/37/T41)-β-Catenin is unaltered in serum-starved Rpgrip1l−/− MEF lysates. Black lines indicate that interven- ing lanes have been spliced out. (D–F, I, and L) Immunofluorescence on MEFs of E12.5 WT and Rpgrip1l−/− embryos (both genotypes: p-β-Catenin: n = 5; p-β-Catenin (3D-SIM, n = 3; Ubiquitin, n = 4; Gli3-190, n = 6; ZsProSensor-1, n = 3; n refers to the number of embryos, respectively). Per embryo, 15 cilia were quantified for p-β-Catenin, 10 cilia were quantified for p-β-Catenin (3D-SIM) and for Ubiquitin, and 20 cilia were quantified for Gli3-190. (G and H) Immunofluorescence on limbs of E12.5 WT and Rpgrip1l−/− embryos (n = 3 embryos, respectively). Per embryo, 20 cilia were quantified for p-β-Catenin and Ubiquitin. All quantified proteins are shown in red (D–J), the ciliary axoneme is marked by acetylated α-tubulin (green; D–J), and the BB is marked by γ-tubulin (blue; D–F, H, and I) or by Pcnt2 (blue; G). (J and K) Immunofluorescence on MEFs of WT embryos (n = 4). 25 cilia per embryo were used for phospho-(S33/37/T41)-β-Catenin and cilia length quantification. (L) Proteasome activity assay on WT and Rpgrip1l−/− MEFs. Cilia are marked by acetylated α-tubulin (α-Tub), and centrosomes/basal bodies are marked by γ-tubulin. Colored squares mark cilia with basal bodies (yellow squares) as well as centrosomes (red squares), which are presented magnified. The green ZsProSensor-1 protein signal is exclusively detected at the ciliary base in Rpgrip1l−/− MEFs. Error bars show standard error of the mean. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 1 µm.

Article Snippet: We used the following primary antibodies: rabbit anti-actin (A2066; Sigma-Aldrich), mouse anti-FLAG (200472; Agilent Technologies), mouse anti-Gapdh (G8795; Sigma-Aldrich), goat anti-Gli3 (AF3690; R&D systems), mouse anti-HA (MMS-101P; Covance), rabbit anti-Hprt (ab10479; Abcam), rabbit anti-Myc (sc-789; Santa Cruz Biotechnology, Inc.), rabbit anti-phospho-(S33/37/T41)-β-Catenin (9561; Cell Signaling Technology), rabbit anti–nonphospho-(S33/37/ T41)-β-Catenin (4270; Cell Signaling Technology), rabbit anti-Psma5 (PA1-1962; Thermo Fisher Scientific), mouse anti-Pcnt (611814; BD), goat anti-Pcnt2 (sc-28145; Santa Cruz Biotechnology, Inc.), goat anti-Psmd2 (IMG-3751; Imgenex), rabbit anti-Psmd3 (S2824; Sigma-Aldrich), rabbit anti-Psmd4 (14899-1-AP; Proteintech), rabbit anti-Smo (ab72130; Abcam), mouse anti–acetylated α-tubulin (T6793; Sigma-Aldrich), mouse anti–γ-tubulin (T6557; Sigma-Aldrich), and rabbit anti-Ubiquitin (U5379; Sigma-Aldrich).

Techniques: Activity Assay, Isolation, Western Blot, Control, Immunofluorescence, Ubiquitin Proteomics

Journal: The Journal of cell biology

Article Title: The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium.

doi: 10.1083/jcb.201408060

Figure Lengend Snippet: Figure 8. Proteasomal activity is unaltered at Rpgrip1l−/− centrosomes. (A–C) Immunofluorescence on MEFs isolated from E12.5 WT and Rpgrip1l−/− embryos (WT: p-β-Catenin (treated with DMSO or MG132): n = 3 embryos; both genotypes: p-β-Catenin and Ubiquitin: n = 3 embryos, respectively). The ciliary axoneme is marked by acetylated α-tubulin (green) and the centrosomes (basal bodies in case of ciliary presence) by γ-tubulin (blue). All quantified proteins are shown in red. An axonemal-like green staining is not visible, demonstrating that the blue staining marks centrosomes. (A) After treatment of WT MEFs with the proteasome inhibitor MG132, the amount of phospho-(S33/37/T41)-β-Catenin is significantly increased at the centrosome. (B and C) The amounts of phospho-(S33/37/T41)-β-Catenin and Ubiquitin are unaltered at the centrosome of Rpgrip1l−/− MEFs. (A–C) Per embryo, 20 cilia were used in the quantifications. Error bars show standard error of the mean. *, P < 0.05. Bars, 0.5 µm.

Article Snippet: We used the following primary antibodies: rabbit anti-actin (A2066; Sigma-Aldrich), mouse anti-FLAG (200472; Agilent Technologies), mouse anti-Gapdh (G8795; Sigma-Aldrich), goat anti-Gli3 (AF3690; R&D systems), mouse anti-HA (MMS-101P; Covance), rabbit anti-Hprt (ab10479; Abcam), rabbit anti-Myc (sc-789; Santa Cruz Biotechnology, Inc.), rabbit anti-phospho-(S33/37/T41)-β-Catenin (9561; Cell Signaling Technology), rabbit anti–nonphospho-(S33/37/ T41)-β-Catenin (4270; Cell Signaling Technology), rabbit anti-Psma5 (PA1-1962; Thermo Fisher Scientific), mouse anti-Pcnt (611814; BD), goat anti-Pcnt2 (sc-28145; Santa Cruz Biotechnology, Inc.), goat anti-Psmd2 (IMG-3751; Imgenex), rabbit anti-Psmd3 (S2824; Sigma-Aldrich), rabbit anti-Psmd4 (14899-1-AP; Proteintech), rabbit anti-Smo (ab72130; Abcam), mouse anti–acetylated α-tubulin (T6793; Sigma-Aldrich), mouse anti–γ-tubulin (T6557; Sigma-Aldrich), and rabbit anti-Ubiquitin (U5379; Sigma-Aldrich).

Techniques: Activity Assay, Immunofluorescence, Isolation, Ubiquitin Proteomics, Staining

Journal: Viruses

Article Title: Rotavirus-Mediated Suppression of miRNA-192 Family and miRNA-181a Activates Wnt/β-Catenin Signaling Pathway: An In Vitro Study

doi: 10.3390/v14030558

Figure Lengend Snippet: RV infection triggers β-Catenin accumulation. Cells infected with RV were harvested at different time points followed by protein expression analysis by Western blotting and Confocal microscopy techniques. ( A ) RV upregulates β-catenin expression during early (3–12 h) hours of infection. Expression of phospho β-catenin Ser37 downregulated in RV infected cells. Expression of phospho β-catenin Ser552 upregulated in RV infected cells; ( B ) Changes in the expression levels of different regulators of the Wnt/β-catenin cellular signaling pathway. Expression of ICAT downregulated in RV infected cells. SMAD4 is activated during RV infection and acts as an attenuator of β-catenin proteasomal degradation. The downstream molecule of β-catenin and SMAD4, TCF1/7 and CCND2 are also activated during RV infection; ( C ) Phosphorylation at Ser552 and destabilization of Ser37 induces β-catenin accumulation in the cytoplasm and nucleus as evident from IF imaging. Scale bars: 50 μm.

Article Snippet: The membranes were then probed with antibodies specific to the proteins phospho-β-catenin ser33/37 (Santa Cruz Biotechnology, Santa Cruz, CA, USA: 57535); phospho-β-catenin ser552 (Cell Signaling Technology, Danvers, MA, USA: 5651); β-catenin (Cell Signaling Technology, Danvers, MA, USA: 8480); ICAT (Santa Cruz Biotechnology: 293489); SMAD4 (Abcam, Waltham, MA, USA: 110175); TCF1/7 (Cell Signaling Technology, Danvers, MA, USA: 2203); CCND2 (Santa Cruz Biotechnology: 56305); Wnt-1 (Santa Cruz Biotechnology: 514531); Wnt-5a (Santa Cruz Biotechnology: 365370); phospho-LRP6 (Cell Signaling Technology, Danvers, MA, USA: 2568); LRP6 (Cell Signaling Technology, Danvers, MA, USA: 3395); phospho-Axin (Merck Millipore, Burlington, MA, USA: ABN1032); Axin (Santa Cruz Biotechnology: 293190); Naked-1 (Cell Signaling Technology, Danvers, MA, USA: 2201), RV-NSP1 (a kind gift from Prof. Koki Taniguchi); RV-NSP4 (prepared according to the standard protocol from Department of Virology and Parasitology, Fujita Health University School of Medicine, Aichi, Japan); and RV- VP6 (HyTest, Turku, Finland: 3C10).

Techniques: Infection, Expressing, Western Blot, Confocal Microscopy, Phospho-proteomics, Imaging

Journal: Viruses

Article Title: Rotavirus-Mediated Suppression of miRNA-192 Family and miRNA-181a Activates Wnt/β-Catenin Signaling Pathway: An In Vitro Study

doi: 10.3390/v14030558

Figure Lengend Snippet: Wnt-1/Wnt-5a pathway activates during RV infection. ( A ) Expression of Wnt-1 and Wnt-5a have been gradually upregulated during RV infection. LRP6 has also been hyper phosphorylated during RV infection; ( B ) Phosphorylation of Axin and its total component have been reduced in RV infected cells. Expression of Nkd1 is suppressed in RV infected cells; ( C ) Phosphorylation of β-catenin Ser552 significantly inhibited in siRNA-FzD9 treated but RV infected cells. The total β-catenin level is also suppressed. The expressions of Wnt-1 and Wnt-5a are also inhibited in siRNA-FzD9 treated but RV infected cells. This may be the result of negative feedback mechanism of Nkd1 or Axin or may be that some other factors are involved in this pathway. The siRNA-FzD9 restricted the RV infection too, as evident by RV-NSP4 expression.

Article Snippet: The membranes were then probed with antibodies specific to the proteins phospho-β-catenin ser33/37 (Santa Cruz Biotechnology, Santa Cruz, CA, USA: 57535); phospho-β-catenin ser552 (Cell Signaling Technology, Danvers, MA, USA: 5651); β-catenin (Cell Signaling Technology, Danvers, MA, USA: 8480); ICAT (Santa Cruz Biotechnology: 293489); SMAD4 (Abcam, Waltham, MA, USA: 110175); TCF1/7 (Cell Signaling Technology, Danvers, MA, USA: 2203); CCND2 (Santa Cruz Biotechnology: 56305); Wnt-1 (Santa Cruz Biotechnology: 514531); Wnt-5a (Santa Cruz Biotechnology: 365370); phospho-LRP6 (Cell Signaling Technology, Danvers, MA, USA: 2568); LRP6 (Cell Signaling Technology, Danvers, MA, USA: 3395); phospho-Axin (Merck Millipore, Burlington, MA, USA: ABN1032); Axin (Santa Cruz Biotechnology: 293190); Naked-1 (Cell Signaling Technology, Danvers, MA, USA: 2201), RV-NSP1 (a kind gift from Prof. Koki Taniguchi); RV-NSP4 (prepared according to the standard protocol from Department of Virology and Parasitology, Fujita Health University School of Medicine, Aichi, Japan); and RV- VP6 (HyTest, Turku, Finland: 3C10).

Techniques: Infection, Expressing, Phospho-proteomics