Journal:

Article Title: Lack of Cyclin-Dependent Kinase 4 Inhibits c- myc Tumorigenic Activities in Epithelial Tissues

doi: 10.1128/MCB.24.17.7538-7547.2004

Figure Lengend Snippet: Western blot analysis of cell cycle proteins from mouse epidermis. Protein lysates of epidermis samples from K5-Myc, K5-Myc/CDK4−/−, K5-Myc/CDK4+/−, CDK4−/−, CDK4+/−, and wild-type siblings were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane. Primary antibodies against CDK4, CDK6, CDK2, cyclin D1, cyclin D2, cyclin A, and cyclin E were used for immunoblot analysis. Protein levels were quantified with a densitometer.

Article Snippet: The following antibodies were used: rabbit polyclonal antibodies against CDK4 (C22), CDK2 (M2), and CDK6 (C21) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.); rabbit polyclonal antibody against cyclin D1 (Ab-3) (Lab Vision Corp./Neo Markers, Fremont, Calif.); and horseradish peroxidase-conjugated secondary antibody (Amersham Corp., Arlington Heights, Ill.).

Techniques: Western Blot, Polyacrylamide Gel Electrophoresis

Journal:

Article Title: Lack of Cyclin-Dependent Kinase 4 Inhibits c- myc Tumorigenic Activities in Epithelial Tissues

doi: 10.1128/MCB.24.17.7538-7547.2004

Figure Lengend Snippet: CDK4 complex formation and kinase assays of K5-Myc epidermis. (A) Complex formation between cyclin D1 and CDK4. Fresh epidermal proteins from K5-Myc and wild-type mice were immunoprecipitated (IP) with a polyclonal antibody against cyclin D1 and immunoblotted with polyclonal antibodies against CDK4 and cyclin D1. Lanes A and B, protein lysates from K5-Myc and wild-type epidermis, respectively. (B) Kinase activity of CDK4 from K5-Myc transgenic and wild-type siblings. Fresh epidermal proteins from two transgenic and two wild-type mice were immunoprecipitated with an anti-CDK4 (IP CDK4) antibody, and in vitro kinase assays were carried out with a pRb peptide as a substrate. (C) Kinase activity of CDK2 from K5-Myc/CDK4+/+, K5-Myc/CDK4+/−, K5-Myc/CDK4−/−, and normal siblings. Fresh epidermal proteins were immunoprecipitated with an anti-CDK2 (IP CDK2) antibody and normal rabbit immunoglobulin G (NR), and in vitro kinase assays were carried out with histone H1 as a substrate.

Article Snippet: The following antibodies were used: rabbit polyclonal antibodies against CDK4 (C22), CDK2 (M2), and CDK6 (C21) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.); rabbit polyclonal antibody against cyclin D1 (Ab-3) (Lab Vision Corp./Neo Markers, Fremont, Calif.); and horseradish peroxidase-conjugated secondary antibody (Amersham Corp., Arlington Heights, Ill.).

Techniques: Immunoprecipitation, Activity Assay, Transgenic Assay, In Vitro

Journal: Scientific Reports

Article Title: Gestational stress disrupts dopamine and oxytocin signaling in the postpartum reward system of rats: implications for mood, motivation and mothering

doi: 10.1038/s41598-024-84043-6

Figure Lengend Snippet: Experimental timeline. Pregnant rats were exposed to a chronic variable stress procedure from gestation day 7 (GD7)-GD20. No stress controls were handled daily. The day of birth (DOB) was designated as postpartum day 0 (PD0). Behavioral testing and brain collection occurred from PD2-PD10. In Expt. 1 , rats (No Stress = 10; Stress = 9) underwent testing for maternal behavior and brains were collected on PD7 for qPCR gene expression analysis of oxytocin (OT) and oxytocin receptor (OTR) in the hypothalamus and ventral tegmental area (VTA). In Expt. 2, rats (No Stress = 6; Stress = 5) were tested on the conditioned place preference (CPP) paradigm. In Expt. 3 , rats (No Stress = 9; Stress = 9) underwent testing on the sucrose preference test (SPT) and brains were collected on PD8 for immunohistochemistry (IHC) and densiometric analysis of dopamine (DA) markers (DAT, dopamine transporter; D1R; dopamine 1 receptor; D2R, dopamine 2 receptor; TH, tyrosine hydroxylase) in the NAc. Maternal and litter characteristics were measured in the SPT dams. In Expt. 4 , rats (No Stress = 12; Stress = 10) were tested on the elevated plus maze (EPM) and forced swim test (FST) and brains were collected on PD10 for IHC and densiometric analysis of additional DA markers in the NAc (pTH, phosphorylated tyrosine hydroxylase; VMAT, vesicular monoamine transporter) as well as counts of TH + cells in the VTA. OT IHC was also done to assess the effects of gestational stress on OT + cells in the hypothalamus and OT fibers in the VTA. In Expt. 5 , dams (No Stress = 6; Stress = 6), the NAc was dissected on PD6 for Liquid Chromatography-Mass Tandem Spectrometry (LC–MS/MS) to measure DA content and the dopamine metabolite, DOPAC. Figure created in https://BioRender.com .

Article Snippet: Next, sections were blocked in 5% BSA in 0.1 M PBS for 1 h at RT and incubated with one of the following primary antibodies: rabbit anti-D1R (1:50; Cat#sc-14001, Santa Cruz Biotechnology), goat anti-dopamine D2R (1:100; Cat#sc-7522, Santa Cruz Biotechnology), rabbit anti-TH (1:1000; Cat#AB152, Millipore), rabbit anti-DAT (1:200; Cat#AB1591P, Millipore), rabbit anti-VMAT(1:100; Cat#PA5-22,864, ThermoFisher), and rabbit anti- pTH (1:500; Cat#AB5935, Millipore) overnight at 4 °C.

Techniques: Gene Expression, Conditioned Place Preference, Immunohistochemistry, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy

Journal: Scientific Reports

Article Title: Gestational stress disrupts dopamine and oxytocin signaling in the postpartum reward system of rats: implications for mood, motivation and mothering

doi: 10.1038/s41598-024-84043-6

Figure Lengend Snippet: Gestational stress alters markers of dopaminergic signaling in the NAc shell. ( A ) In the nucleus accumbens (NAc) shell, markers associated with dopaminergic signaling were examined via immunohistochemistry on PD8 or PD10. Compared to unstressed controls, dams exposed to gestational stress showed a reduction in percent area staining for several dopaminergic markers including ( B ) tyrosine hydroxylase, TH, ( C ) phosphorylated TH, pTH and ( D ) the dopamine transporter, DAT. ( E ) There was no effect of gestational stress on vesicular monoamine transporter, VMAT. Percent area staining for dopamine receptors ( F ) D1R and ( G ) D2R in the NAc shell was also examined and only the D2R was reduced by gestational stress. Representative images of each marker analyzed are shown in ( A ). * p < 0.05; ** p < 0.005; *** p ≤ 0.0005.

Article Snippet: Next, sections were blocked in 5% BSA in 0.1 M PBS for 1 h at RT and incubated with one of the following primary antibodies: rabbit anti-D1R (1:50; Cat#sc-14001, Santa Cruz Biotechnology), goat anti-dopamine D2R (1:100; Cat#sc-7522, Santa Cruz Biotechnology), rabbit anti-TH (1:1000; Cat#AB152, Millipore), rabbit anti-DAT (1:200; Cat#AB1591P, Millipore), rabbit anti-VMAT(1:100; Cat#PA5-22,864, ThermoFisher), and rabbit anti- pTH (1:500; Cat#AB5935, Millipore) overnight at 4 °C.

Techniques: Immunohistochemistry, Staining, Marker

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase HDAC2 silencing prevents endometriosis by activating the HNF4A/ARID1A axis

doi: 10.1111/jcmm.16835

Figure Lengend Snippet: HDAC2 expression is high in endometriosis. (A) The volcano map of gene expression in GSE37837 . The black dot indicates the genes with no significant difference, the red dot indicates the significantly upregulated gene, and the green dot indicates the significantly downregulated gene. (B) Venn map of 40 intersected genes between the significantly upregulated genes of GSE37837 and the gene retrieved by GeneCards. (C) The importance of genes in endometriosis analysed by phenolyzer. When the length of the column was longer, the importance was higher. (D) HDAC2 expression in GSE37837 . The left blue box showed the expression in normal samples, and the right red box showed the expression in endometriosis samples. The data were expressed as median ± interquartile range. (E) GEPIA of the expression of HDAC2 in UCEC. The left red box was the expression in UCEC, and the right grey box was the expression in the control group. The data were expressed as median ± interquartile range. * p < 0.05. (F) The expression of HDAC2 protein in normal tissues ( n = 30) and endometriosis tissues ( n = 40) detected by immunohistochemistry. The data were expressed as median ± interquartile range. (G) Western blot analysis of the expression of HDAC2 protein in normal tissues ( n = 30) and endometriosis tissues ( n = 40). The data were expressed as median ± interquartile range. * p < 0.05 vs. the control group. The experiment was repeated three times independently

Article Snippet: Primary rabbit anti‐human antibodies to HDAC2 (12922‐3‐AP, 1:200, Proteintech), HNF4A (ab92378, 1:500, Abcam), Ki‐67 (ab16667, 1:200, Abcam), and ARID1A (ab182560, 1:1000, Abcam) were added to the sections for overnight culture at 4°C, and the secondary antibody (ab6785, 1:1000, Abcam) was added.

Techniques: Expressing, Gene Expression, Control, Immunohistochemistry, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase HDAC2 silencing prevents endometriosis by activating the HNF4A/ARID1A axis

doi: 10.1111/jcmm.16835

Figure Lengend Snippet: Silencing HDAC2 decreases the proliferation and invasion and increases the apoptosis of endometriosis cells. (A) The expression of HDAC2 in hEM15A cells of each group detected by Western blot analysis. (B) The proliferation of hEM15A cells in each group detected by CCK‐8 method. (C) The invasion of hEM15A cells in each group measured by Transwell assay. (D) The apoptosis of hEM15A cells in each group detected by flow cytometry. Data were shown as median ± standard error of mean ( n = 3). * p < 0.05 vs. the sh‐NC group. The experiment was repeated three times independently

Article Snippet: Primary rabbit anti‐human antibodies to HDAC2 (12922‐3‐AP, 1:200, Proteintech), HNF4A (ab92378, 1:500, Abcam), Ki‐67 (ab16667, 1:200, Abcam), and ARID1A (ab182560, 1:1000, Abcam) were added to the sections for overnight culture at 4°C, and the secondary antibody (ab6785, 1:1000, Abcam) was added.

Techniques: Expressing, Western Blot, CCK-8 Assay, Transwell Assay, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase HDAC2 silencing prevents endometriosis by activating the HNF4A/ARID1A axis

doi: 10.1111/jcmm.16835

Figure Lengend Snippet: HDAC2 reduces HNF4A expression through deacetylation. (A) Pearson correlation map of HDAC2 and HNF4A expression in GSE37837 . (B) The expression of HNF4A protein in normal tissues ( n = 30) and endometriosis tissues ( n = 40) determined by immunohistochemistry. The data were expressed as median ± interquartile range. (C) The expression of HNF4A protein in normal tissues ( n = 30) and endometriosis tissues ( n = 40) measured by Western blot analysis. The data were expressed as median ± interquartile range. (D) The enrichment of HDAC2 in the promoter region of HNF4A in normal tissues ( n = 30) and endometriosis tissues ( n = 40) measured by ChIP. (E) The enrichment of HDAC2 in the promoter region of HNF4A of each group detected by ChIP. (F) The expression of HDAC2 and HNF4A in each group assessed by Western blot analysis. (G) HNF4A acetylation level following IP. Data were shown as median ± standard error of mean ( n = 6). * p < 0.05 vs. the sh‐NC group; # p < 0.05 vs. the oe‐NC group. The experiment was repeated three times independently

Article Snippet: Primary rabbit anti‐human antibodies to HDAC2 (12922‐3‐AP, 1:200, Proteintech), HNF4A (ab92378, 1:500, Abcam), Ki‐67 (ab16667, 1:200, Abcam), and ARID1A (ab182560, 1:1000, Abcam) were added to the sections for overnight culture at 4°C, and the secondary antibody (ab6785, 1:1000, Abcam) was added.

Techniques: Expressing, Immunohistochemistry, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase HDAC2 silencing prevents endometriosis by activating the HNF4A/ARID1A axis

doi: 10.1111/jcmm.16835

Figure Lengend Snippet: HDAC2 silencing elevates HNF4A expression to slow the proliferation and invasion and facilitate the apoptosis of endometriosis cells. (A) The expression of HDAC2 and HNF4A in hEM15A cells of each group evaluated by Western blot analysis. (B) The viability of hEM15A cells determined by CCK‐8 method. (C) hEM15A cell invasion in each group measured by Transwell assay. (D) Apoptosis of hEM15A cells in each group detected by flow cytometry. Data were shown as median ± standard error of mean ( n = 3). * p < 0.05 vs. the sh‐NC + sh‐NC group; # p < 0.05 vs. the sh‐HDAC2 + sh‐NC group. The experiment was repeated three times independently

Article Snippet: Primary rabbit anti‐human antibodies to HDAC2 (12922‐3‐AP, 1:200, Proteintech), HNF4A (ab92378, 1:500, Abcam), Ki‐67 (ab16667, 1:200, Abcam), and ARID1A (ab182560, 1:1000, Abcam) were added to the sections for overnight culture at 4°C, and the secondary antibody (ab6785, 1:1000, Abcam) was added.

Techniques: Expressing, Western Blot, CCK-8 Assay, Transwell Assay, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase HDAC2 silencing prevents endometriosis by activating the HNF4A/ARID1A axis

doi: 10.1111/jcmm.16835

Figure Lengend Snippet: Endometriosis is repressed in vivo by silencing HDAC2. (A) The area of endometriosis tissues of mice in each group. (B) Endometriosis tissue weight of mice in each group. (C) The expression of HDAC2, HNF4A and ARID1A in endometriosis tissues of each group analysed by immunohistochemistry. (D) Ki‐67 expression measured by immunohistochemistry and apoptosis detected by TUNEL staining in each group. Data were shown as median ± standard error of mean ( n = 8). * p < 0.05 vs. the sh‐NC group. n = 8 mice/group. The experiment was repeated three times independently

Article Snippet: Primary rabbit anti‐human antibodies to HDAC2 (12922‐3‐AP, 1:200, Proteintech), HNF4A (ab92378, 1:500, Abcam), Ki‐67 (ab16667, 1:200, Abcam), and ARID1A (ab182560, 1:1000, Abcam) were added to the sections for overnight culture at 4°C, and the secondary antibody (ab6785, 1:1000, Abcam) was added.

Techniques: In Vivo, Expressing, Immunohistochemistry, TUNEL Assay, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase HDAC2 silencing prevents endometriosis by activating the HNF4A/ARID1A axis

doi: 10.1111/jcmm.16835

Figure Lengend Snippet: Molecular mechanism of the HDAC2/HNF4A/ARID1A axis involved in endometriosis. HDAC2 inhibited HNF4A through deacetylation, thus diminishing ARID1A expression to promote endometriosis

Article Snippet: Primary rabbit anti‐human antibodies to HDAC2 (12922‐3‐AP, 1:200, Proteintech), HNF4A (ab92378, 1:500, Abcam), Ki‐67 (ab16667, 1:200, Abcam), and ARID1A (ab182560, 1:1000, Abcam) were added to the sections for overnight culture at 4°C, and the secondary antibody (ab6785, 1:1000, Abcam) was added.

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: Parkin ubiquitinates phosphoglycerate dehydrogenase to suppress serine synthesis and tumor progression

doi: 10.1172/JCI132876

Figure Lengend Snippet: (A) PHGDH-Flag interacted with Myc-Parkin in MCF10A cells. Cells with ectopic expression of PHGDH-Flag and Myc-Parkin were employed for co-IP assays using the anti-Flag (left) and anti-Myc antibodies (right), respectively. (B and C) Endogenous PHGDH interacted with endogenous Parkin in Hs578T (B) and H1299 cells (C), as detected by co-IP assays. PHGDH was knocked down by shRNAs in cells as negative controls. (D) Co-IP analysis of interaction of endogenous PHGDH and Parkin in WT Hs578T cells and Hs578T cells with PHGDH KO by CRISPR/Cas9. (E) Parkin bound to PHGDH at its SBD2 domain. Left: schematic representation of vectors expressing WT or serial deletion mutants of PHGDH-Flag. (F) PHGDH bound to Parkin at its IBR domain. Left: schematic representation of vectors expressing WT or serial deletion mutants of Myc-Parkin. (G) Direct interaction of recombinant GST-Parkin and His-Trx-PHGDH proteins analyzed by in vitro GST pull-down assays.

Article Snippet: The levels of ubiquitination of endogenous PHGDH were determined by IP of PHGDH using an anti-PHGDH antibody (catalog 66350, Cell Signaling Technology) followed by Western blot assays using an anti-HA antibody (catalog 11867423001, Sigma-Aldrich, 1:1000).

Techniques: Expressing, Co-Immunoprecipitation Assay, CRISPR, Recombinant, In Vitro

Journal: The Journal of Clinical Investigation

Article Title: Parkin ubiquitinates phosphoglycerate dehydrogenase to suppress serine synthesis and tumor progression

doi: 10.1172/JCI132876

Figure Lengend Snippet: (A) Myc-Parkin expression reduced levels of PHGDH-Flag protein in Hs578T and H1299 cells. Cells were transfected with the PHGDH-Flag vector together with varying amounts of Myc-Parkin or control (Con) vectors. (B) Ectopic Myc-Parkin expression reduced levels of endogenous PHGDH protein in different human breast and lung cancer cells. (C) Knockdown of endogenous Parkin by 2 different shRNA vectors increased levels of endogenous PHGDH protein in different human breast and lung cells. (D) KO of Parkin by CRISPR/Cas9 increased levels of PHGDH protein in Hs578T and H1299 cells. (E) Higher PHGDH protein levels in Parkin−/− MEFs compared with Parkin+/+ MEFs. (F) Higher PHGDH protein levels in the breast and lung tissues of Parkin–/– mice compared with Parkin+/+ mice. n = 5 mice/group.

Article Snippet: The levels of ubiquitination of endogenous PHGDH were determined by IP of PHGDH using an anti-PHGDH antibody (catalog 66350, Cell Signaling Technology) followed by Western blot assays using an anti-HA antibody (catalog 11867423001, Sigma-Aldrich, 1:1000).

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

Journal: The Journal of Clinical Investigation

Article Title: Parkin ubiquitinates phosphoglycerate dehydrogenase to suppress serine synthesis and tumor progression

doi: 10.1172/JCI132876

Figure Lengend Snippet: (A) Treatment of proteasome inhibitor MG132 increased PHGDH protein levels and abolished inhibitory effect of Parkin on PHGDH levels in Hs578T cells. Hs578T cells with ectopic expression of WT or C431A Myc-Parkin (upper) and WT or Parkin-KO Hs578T cells (lower) were treated with MG132 (5 μM) or DMSO for 12 hours before Western blot assays. (B) Myc-Parkin expression decreased PHGDH protein half-life (left), whereas knockdown of endogenous Parkin increased PHGDH protein half-life (right) in Hs578T cells. Cells were treated with CHX or DMSO for different hours before Western blot assays. (C) KO of Parkin by CRISPR/Cas9 increased PHGDH protein half-life in Hs578T cells. In B and C, data are presented as mean ± SD. n = 3. (D) Effects of WT and mutant Myc-Parkin on ubiquitination of PHGDH-Flag in Hs578T cells analyzed by in vivo ubiquitination (Ub) assays. (E) GST-Parkin ubiquitinates His-Trx-PHGDH in vitro analyzed by in vitro ubiquitination assays using recombinant proteins. (F) Parkin knockdown by shRNA (left) or Parkin KO by CRISPR/Cas9 (middle and right) reduced ubiquitination of PHGDH-Flag in Hs578T and H1299 cells analyzed by in vivo ubiquitination assays. (G) Parkin knockdown by shRNA reduced ubiquitination of endogenous PHGDH in Hs578T cells analyzed by in vivo ubiquitination assays. (H) T240M and P294S mutations impaired Parkin’s ubiquitination activity toward PHGDH in Hs578T cells analyzed by in vivo ubiquitination assays. (I) T240M and P294S mutations impaired the ability of Myc-Parkin to downregulate PHGDH protein levels in Hs578T and H1299 cells.

Article Snippet: The levels of ubiquitination of endogenous PHGDH were determined by IP of PHGDH using an anti-PHGDH antibody (catalog 66350, Cell Signaling Technology) followed by Western blot assays using an anti-HA antibody (catalog 11867423001, Sigma-Aldrich, 1:1000).

Techniques: Expressing, Western Blot, CRISPR, Mutagenesis, In Vivo, In Vitro, Recombinant, shRNA, Activity Assay

Journal: The Journal of Clinical Investigation

Article Title: Parkin ubiquitinates phosphoglycerate dehydrogenase to suppress serine synthesis and tumor progression

doi: 10.1172/JCI132876

Figure Lengend Snippet: (A) Top 3 potential lysine ubiquitination sites in PHGDH identified by LC-MS/MS analysis, including K310, K330, and K364. (B) K330 mutation (K330R) largely abolished ubiquitination of PHGDH by Parkin. Hs578T cells with expression of WT or indicated mutant PHGDH-Flag were used for in vivo ubiquitination assays. (C) K330R mutation largely abolished the negative regulation of PHGDH protein levels by Myc-Parkin in Hs578T and H1299 cells. Hs578T and H1299 cells were transduced with WT or K330R PHGDH-Flag vectors together with Myc-Parkin vectors for Western blot assays. (D) Myc-Parkin expression did not clearly affect the K330R PHGDH-Flag protein half-life in Hs578T cells. Data are presented as mean ± SD. n = 3.

Article Snippet: The levels of ubiquitination of endogenous PHGDH were determined by IP of PHGDH using an anti-PHGDH antibody (catalog 66350, Cell Signaling Technology) followed by Western blot assays using an anti-HA antibody (catalog 11867423001, Sigma-Aldrich, 1:1000).

Techniques: Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Expressing, In Vivo, Transduction, Western Blot

Journal: Cell

Article Title: Local Translation of Extranuclear Lamin B Promotes Axon Maintenance

doi: 10.1016/j.cell.2011.11.064

Figure Lengend Snippet: The Level of LB2 Protein in Retinal Cultures Is Increased by BDNF Stimulation, Related to Figure 1 (A) One 2D-DIGE spot (out of ∼1,300 putative spots) that increased in intensity following 24 hr BDNF stimulation of retinal cultures (arrow). (B) Biological variation analysis (BVA) of 2D-DIGE (mean ± SEM; 3 replicates; ∗∗∗ p < 0.001; unpaired t test). MS analysis identified the spot as LB2 ( Xenopus laevis ; Gene name— lmnb2 ; MASCOT score-66).

Article Snippet: Immunofluorescence Immunostaining, imaging, and analysis were performed as described ( ) using the following antibodies: anti-LB2 antibodies (Ab1—mouse monoclonal clone LN43, 1:100, Abcam; Ab2—rabbit polyclonal, 1:200, ab8983, Abcam; Ab3- mouse monoclonal clone X223, 1:100, Santa Cruz Biotechnology); rabbit polyclonal anti-CoxIV antibody (1:100, Abcam); goat polyclonal VDAC2 antibody (1:100, Abcam); and mouse monoclonal β-actin antibody (1:400, Abcam).

Techniques:

Journal: Cell

Article Title: Local Translation of Extranuclear Lamin B Promotes Axon Maintenance

doi: 10.1016/j.cell.2011.11.064

Figure Lengend Snippet: En-1 Stimulation Elicits Dynamic Changes in Local Protein Synthesis (A) Axon culture preparation by separating the eye. The absence of DNA in the axonal fraction confirms its purity. (B) DIGE-NCAT detection of newly synthesized axonal polypeptides shows AHA incorporation in spots corresponding to LB2 (red: control; green: En-1; and arrows: LB2 spot). (C and D) Quantitative analysis of putative protein spots between control and En-1 conditions (green: increased; red: decreased; and yellow: unchanged). See also Figure S2 and .

Article Snippet: Immunofluorescence Immunostaining, imaging, and analysis were performed as described ( ) using the following antibodies: anti-LB2 antibodies (Ab1—mouse monoclonal clone LN43, 1:100, Abcam; Ab2—rabbit polyclonal, 1:200, ab8983, Abcam; Ab3- mouse monoclonal clone X223, 1:100, Santa Cruz Biotechnology); rabbit polyclonal anti-CoxIV antibody (1:100, Abcam); goat polyclonal VDAC2 antibody (1:100, Abcam); and mouse monoclonal β-actin antibody (1:400, Abcam).

Techniques: Synthesized, Control

Journal: Cell

Article Title: Local Translation of Extranuclear Lamin B Promotes Axon Maintenance

doi: 10.1016/j.cell.2011.11.064

Figure Lengend Snippet: LB2 mRNA and Protein Are Expressed in RGC Axons and GCs (A) Axonal lb2 mRNA detected by RT-PCR. (B and C) Lb2 FISH in cultured retinal axons and its quantitation (mean ± SEM; n = no. of GC; ∗ p < 0.05; Mann-Whitney). (D–G′) ISH of stage 40–45 embryo sections (RGC: retinal ganglion cell layer; ONH: optic nerve head; and IPL/OPL: inner/outer plexiform layer). The same sections were counterstained for neurofilament and DAPI. (H and I) LB2 immunostaining in cultured retinal axons and its reduction by LB2MO. (J and K′) Immunostaining in tissue sections. The boxed areas are shown in the lower panels. Scale bars, 5 μm in (B) and (H), 25 μm in (D)–(G), (J), and (K), and 10 μm in (J) and (K) lower panels. See also Figure S3 .

Article Snippet: Immunofluorescence Immunostaining, imaging, and analysis were performed as described ( ) using the following antibodies: anti-LB2 antibodies (Ab1—mouse monoclonal clone LN43, 1:100, Abcam; Ab2—rabbit polyclonal, 1:200, ab8983, Abcam; Ab3- mouse monoclonal clone X223, 1:100, Santa Cruz Biotechnology); rabbit polyclonal anti-CoxIV antibody (1:100, Abcam); goat polyclonal VDAC2 antibody (1:100, Abcam); and mouse monoclonal β-actin antibody (1:400, Abcam).

Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Quantitation Assay, MANN-WHITNEY, Immunostaining

Journal: Cell

Article Title: Local Translation of Extranuclear Lamin B Promotes Axon Maintenance

doi: 10.1016/j.cell.2011.11.064

Figure Lengend Snippet: Lb2 mRNA and LB2 Protein Localize to Axons, Related to Figure 3 (A and B) Lb2 and pax6 ISH in eye sections (RGC: retinal ganglion cell layer; ONH: optic nerve head; ON: optic nerve). The same sections were counterstained for axonal (neurofilament) and nuclear (DAPI) markers. The ONH, where RGC axons collect to exit the eye, is devoid of cell bodies and contains lb2 mRNA but not pax6 mRNA. (C and D) Lb2 and pax6 ISH in brain sections with a counterstained image. The tectal neuropil, where RGC axons terminate, is devoid of cell bodies and contains lb2 mRNA but not pax6 mRNA (e.g., inside the red dashed line). (E) LB2 immunostaining in brain sections shows that LB2 localizes to RGC axons in the optic tectum (Tec) (e.g., inside the red dashed line). Arrowheads indicate axons ascending to the optic tectum. (F and G) Brn3, a nuclear factor abundantly expressed in RGCs, does not localize to the tectal neuropil (e.g., inside the red dashed line) or nearby tegmental neuropil (white arrow), although it is highly expressed by a subset of neurons in the tegmentum (green arrow). (H) LB2 antibody recognizes the nucleus (red) as expected (two left panels) in fibroblast-like cells in eye explant culture. Increasing the exposure reveals localization of LB2 outside the nucleus (upper right panel). LB2MO significantly decreases the nuclear LB2 staining (arrowhead), examined at the same exposure (two middle panels). Increasing the exposure shows that the extranuclear LB2 staining (arrow) is also reduced with LB2MO, indicating that the cytoplasmic LB2 signal is specific (two right panels). (I) Western blot of stage 40 embryo head lysate shows that LB2MO inhibits LB2 translation in a dose-dependent manner. (J) Quantification of the lane intensity of LB2/α-tubulin for the indicated conditions. (K and L) Two additional independent antibodies against LB2 detect a single major band in western blot and extranuclear LB2 in axons and GCs in culture. (M) LB2MO reduces axonal LB2 detected by a different antibody confirming the specificity of this signal ( ∗∗ p < 0.01; unpaired t test). Scale bars: 25 μm in (A)–(E), 5 μm in (F) and (J).

Article Snippet: Immunofluorescence Immunostaining, imaging, and analysis were performed as described ( ) using the following antibodies: anti-LB2 antibodies (Ab1—mouse monoclonal clone LN43, 1:100, Abcam; Ab2—rabbit polyclonal, 1:200, ab8983, Abcam; Ab3- mouse monoclonal clone X223, 1:100, Santa Cruz Biotechnology); rabbit polyclonal anti-CoxIV antibody (1:100, Abcam); goat polyclonal VDAC2 antibody (1:100, Abcam); and mouse monoclonal β-actin antibody (1:400, Abcam).

Techniques: Immunostaining, Staining, Western Blot

Journal: Cell

Article Title: Local Translation of Extranuclear Lamin B Promotes Axon Maintenance

doi: 10.1016/j.cell.2011.11.064

Figure Lengend Snippet: LB2 Is Locally Synthesized In Vitro and In Vivo (A–C) LB2 QIF from axon-only culture (mean ± SEM; n = no. of GCs; 3 replicates; ∗∗ p < 0.01; one-way ANOVA and Bonferroni). (D) Axon-TRAP experiment. GFP-L10a RNA is expressed by blastomere injections in the CNS of a donor embryo, whose eye is transplanted into an uninjected host. The transplanted eye then extends retinal axons to the contralateral optic tectum of the host brain. The third diagram represents a brain that has been cut at the ventral midline and flattened. The boxed areas were dissected out, from which GFP-L10a-containing ribosomes (green) and associated mRNAs were purified by GFP immunoprecipitation. In the negative control, GFP RNA was used instead of L10a-GFP RNA . (E) RT-PCR from axon-TRAP for β-actin mRNA (TI: total input; IP: immunoprecipitation). (F) RT-PCR for lb2 mRNA. Scale bar, 4 μm. See also Figure S4 .

Article Snippet: Immunofluorescence Immunostaining, imaging, and analysis were performed as described ( ) using the following antibodies: anti-LB2 antibodies (Ab1—mouse monoclonal clone LN43, 1:100, Abcam; Ab2—rabbit polyclonal, 1:200, ab8983, Abcam; Ab3- mouse monoclonal clone X223, 1:100, Santa Cruz Biotechnology); rabbit polyclonal anti-CoxIV antibody (1:100, Abcam); goat polyclonal VDAC2 antibody (1:100, Abcam); and mouse monoclonal β-actin antibody (1:400, Abcam).

Techniques: Synthesized, In Vitro, In Vivo, Purification, Immunoprecipitation, Negative Control, Reverse Transcription Polymerase Chain Reaction

Journal: Cell

Article Title: Local Translation of Extranuclear Lamin B Promotes Axon Maintenance

doi: 10.1016/j.cell.2011.11.064

Figure Lengend Snippet: Axonal Translation of lb2 mRNA Is Selectively Regulated, Related to Figure 4 (A–C) One hour En-1 stimulation does not induce translation of β-actin mRNA, which is localized to RGC axons. ns: not significant; unpaired t test. (D) Lb2 mRNA is expressed at a similar level in the eye to other mRNAs encoding nuclear proteins as revealed by quantitatve RT-PCR. (E) Axon-TRAP analysis shows that other lamin mRNAs are not associated with ribosomes in RGC axons in vivo. Scale bar, 5 μm.

Article Snippet: Immunofluorescence Immunostaining, imaging, and analysis were performed as described ( ) using the following antibodies: anti-LB2 antibodies (Ab1—mouse monoclonal clone LN43, 1:100, Abcam; Ab2—rabbit polyclonal, 1:200, ab8983, Abcam; Ab3- mouse monoclonal clone X223, 1:100, Santa Cruz Biotechnology); rabbit polyclonal anti-CoxIV antibody (1:100, Abcam); goat polyclonal VDAC2 antibody (1:100, Abcam); and mouse monoclonal β-actin antibody (1:400, Abcam).

Techniques: Reverse Transcription Polymerase Chain Reaction, In Vivo

Journal: Cell

Article Title: Local Translation of Extranuclear Lamin B Promotes Axon Maintenance

doi: 10.1016/j.cell.2011.11.064

Figure Lengend Snippet: Inhibiting LB2 Translation Results in Degeneration of RGC Pathway In Vivo (A) MO injection and RGC axon labeling using DiI (green: MO; Tel: telencephalon; Ch: optic chiasm; OT: optic tract; Tec: optic tectum; and red: DiI). (B) DiI-labeled RGC axons in MO-injected embryos with or without LB2-GFP RNA. (C) Characteristic beaded morphology of dying axons in LB2 morphants. The boxed area is shown in the right panel. (D) Quantitative analysis of the LB2MO-induced reduction in RGC axons and its rescue by LB2-GFP (n = no. of brains; 3 replicates; ∗∗∗∗ p < 0.0001; Fisher's exact). (E and F) The frequency of active caspase-3-positive cells in retinal sections (mean ± SEM; n = sections analyzed; Mann-Whitney). Scale bars: 65 μm in (B) and (C), 100 μm in (C) right panel, and 25 μm in (E). See also Figure S5 .

Article Snippet: Immunofluorescence Immunostaining, imaging, and analysis were performed as described ( ) using the following antibodies: anti-LB2 antibodies (Ab1—mouse monoclonal clone LN43, 1:100, Abcam; Ab2—rabbit polyclonal, 1:200, ab8983, Abcam; Ab3- mouse monoclonal clone X223, 1:100, Santa Cruz Biotechnology); rabbit polyclonal anti-CoxIV antibody (1:100, Abcam); goat polyclonal VDAC2 antibody (1:100, Abcam); and mouse monoclonal β-actin antibody (1:400, Abcam).

Techniques: In Vivo, Injection, Labeling, MANN-WHITNEY

Journal: Cell

Article Title: Local Translation of Extranuclear Lamin B Promotes Axon Maintenance

doi: 10.1016/j.cell.2011.11.064

Figure Lengend Snippet: LB2MO-Induced Pathway Degeneration Is Mainly Axonal and Results from Inhibition of Local LB2 Synthesis (A) Eye electroporation. (B) DiI-labeled RGC axons in eye-electroporated embryos. (C and D) The mean DiI intensity of the distal 150 μm of the RGC pathway (mean ± SEM; n = no. of brains; 3 replicates; ∗∗∗∗ p < 0.0001; Mann-Whitney). (E) Pathway electroporation. (F and G) DiI-labeled RGC axons in pathway-electroporated embryos (mean ± SEM; n = no. of brains; 3 replicates; ∗∗ p < 0.01; Fisher's exact). (H) Nuclear LB2 immunofluorescent intensity in the RGC layer normalized to its intensity in the INL (mean ± SEM; for CoMO, total RGC no. = 1648, total INL cell no. = 2813, total no. of sections = 15; for LB2MO, total RGC no. = 1639, total INL cell no. = 3001, total no. of sections = 14; Mann-Whitney). (I) Wild-type LB2 and LB2ΔNLS localization in HEK293T cells (arrowhead: nuclear; arrow: cytoplasmic). (J) Rescue experiment. (K and L) Ath5:RFP-labeled RGC axons in LB2MO-injected embryos with or without LB2ΔNLS eye electroporation. Tel: telencephalon; Ch: optic chiasm; OT: optic tract; Tec: optic tectum. Scale bars: 65 μm in (B), (F), and (K) and 10 μm in (I). See also Figure S6 .

Article Snippet: Immunofluorescence Immunostaining, imaging, and analysis were performed as described ( ) using the following antibodies: anti-LB2 antibodies (Ab1—mouse monoclonal clone LN43, 1:100, Abcam; Ab2—rabbit polyclonal, 1:200, ab8983, Abcam; Ab3- mouse monoclonal clone X223, 1:100, Santa Cruz Biotechnology); rabbit polyclonal anti-CoxIV antibody (1:100, Abcam); goat polyclonal VDAC2 antibody (1:100, Abcam); and mouse monoclonal β-actin antibody (1:400, Abcam).

Techniques: Inhibition, Electroporation, Labeling, MANN-WHITNEY, Injection

Journal: Cell

Article Title: Local Translation of Extranuclear Lamin B Promotes Axon Maintenance

doi: 10.1016/j.cell.2011.11.064

Figure Lengend Snippet: Axonally Synthesized LB2 Localizes to Mitochondria, Related to Figure 6 (A) Detailed schematic representation of pathway electroporation described in Figure 6 E. At stage 40, one side of the optic tectum is exposed by removing the eye and skin before MO is electroporated. (B–D) HEK293T cells transfected with Xenopus LB2ΔNLS plasmid were labeled with MitoTracker, and imaged by a laser-scanning confocal microscope. LB2ΔNLS does not localize to the nuclear membrane (arrowhead) and instead localize to cytoplasmic structures that include mitochondria (arrow). Cross-sectional views seen from the dashed lines in (D) are shown left and above each image. (E–G) LB2ΔNLS also localizes to mitochondria in cultured Xenopus RGC axons (arrow). (H) In contrast, wild-type LB2 mainly localizes to the nuclear membrane. Scale bars: (B–D) and (H), 10 μm; (E–G), 5 μm.

Article Snippet: Immunofluorescence Immunostaining, imaging, and analysis were performed as described ( ) using the following antibodies: anti-LB2 antibodies (Ab1—mouse monoclonal clone LN43, 1:100, Abcam; Ab2—rabbit polyclonal, 1:200, ab8983, Abcam; Ab3- mouse monoclonal clone X223, 1:100, Santa Cruz Biotechnology); rabbit polyclonal anti-CoxIV antibody (1:100, Abcam); goat polyclonal VDAC2 antibody (1:100, Abcam); and mouse monoclonal β-actin antibody (1:400, Abcam).

Techniques: Synthesized, Electroporation, Transfection, Plasmid Preparation, Labeling, Microscopy, Membrane, Cell Culture

Journal: Cell

Article Title: Local Translation of Extranuclear Lamin B Promotes Axon Maintenance

doi: 10.1016/j.cell.2011.11.064

Figure Lengend Snippet: LB2 Knockdown Interferes with Mitochondrial Functions in Axons (A and B) LB2 coimmunostaining with CoxIV and VDAC2. (C) A single plane OMX super-resolution image of LB2 and Mito-GFP. (D) TMRM staining of CoMO- or LB2MO-injected axons and GCs. (E) Mitochondrial potential (Fm/Fc) in the distal 30 μm GCs/axons (mean ± SEM; n = no. of mitochondria analyzed; 3 replicates; ∗∗∗ p < 0.0001; Mann-Whitney). (F and G) Mitochondrial length (box-and-whisker plot: minimum and maximum) ( ∗∗∗ p < 0.0001; Mann-Whitney). (H) Mitochondrial number per unit length axon (box-and-whisker plot) (n.s.: not significant). (I) Anterograde and retrograde organelle transport measured by lysosome movements (mean ± SEM; 18 CoMO axons and 15 LB2MO axons; ∗∗ p < 0.01 and ∗∗∗ p < 0.001; Dunn's multiple comparison and Kruskal-Wallis). Scale bars: 1 μm in (C) and 5 μm in the rest. See also Figure S7 .

Article Snippet: Immunofluorescence Immunostaining, imaging, and analysis were performed as described ( ) using the following antibodies: anti-LB2 antibodies (Ab1—mouse monoclonal clone LN43, 1:100, Abcam; Ab2—rabbit polyclonal, 1:200, ab8983, Abcam; Ab3- mouse monoclonal clone X223, 1:100, Santa Cruz Biotechnology); rabbit polyclonal anti-CoxIV antibody (1:100, Abcam); goat polyclonal VDAC2 antibody (1:100, Abcam); and mouse monoclonal β-actin antibody (1:400, Abcam).

Techniques: Knockdown, Staining, Injection, MANN-WHITNEY, Whisker Assay, Comparison

Journal: Cell

Article Title: Local Translation of Extranuclear Lamin B Promotes Axon Maintenance

doi: 10.1016/j.cell.2011.11.064

Figure Lengend Snippet: LB2 Localizes to Mitochondria, Related to Figure 7 (A) LB2 and Nudel show a relatively weak colocalization. (B) LB2 detected by a second LB2 antibody colocalizes with VDAC2, a mitochondrial protein. (C) Schematic representation of PLA technology. PLA signal from fluorescent oligonucleotides represents proximity of the two proteins of interest (within 40 nm). (D) Representative images of cultured RGC GCs, showing PLA signals obtained with indicated antibody pairs. LB2 interacts with CoxIV, but not with NFPC or neuropilin1. Positive controls (the last two panels) show specific signals within the GC, which is consistent with reported protein-protein interactions. Scale bars, 5 μm.

Article Snippet: Immunofluorescence Immunostaining, imaging, and analysis were performed as described ( ) using the following antibodies: anti-LB2 antibodies (Ab1—mouse monoclonal clone LN43, 1:100, Abcam; Ab2—rabbit polyclonal, 1:200, ab8983, Abcam; Ab3- mouse monoclonal clone X223, 1:100, Santa Cruz Biotechnology); rabbit polyclonal anti-CoxIV antibody (1:100, Abcam); goat polyclonal VDAC2 antibody (1:100, Abcam); and mouse monoclonal β-actin antibody (1:400, Abcam).

Techniques: Cell Culture, Protein-Protein interactions