rabbit polyclonal prmt5 antibody  (EpiGentek)

Journal: bioRxiv

Article Title: The E3 ligase NEDD4L prevents colorectal cancer liver metastasis via degradation of PRMT5 to inhibit the AKT/mTOR signaling pathway

doi: 10.1101/2024.10.21.619337

Figure Lengend Snippet: NEDD4L ubiquitinates PRMT5 to promote its degradation. ( A) EZBlue™ staining of gels containing the total lysate of 293FT cells overexpressing Flag-NEDD4L and HA-ubiquitin. The immunoprecipitates obtained from an immunoprecipitation assay of 293FT cells with overexpression of Flag-NEDD4L and HA-ubiquitin were incubated with 20 μM MG132 for 12 hr and pulled down by Flag or Flag and HA agarose. ( B) Representative western blots of proteins immunoprecipitated from 293FT cells overexpressing Flag-NEDD4L and HA-ubiquitin overexpression that were incubated with 20 μM MG132 for 12 hr and pulled down by Flag or Flag and HA agarose. Three independent western blot analyses were performed. ( C) Venn diagram showing the overlap among the candidate proteins identified by mass spectrometry analysis of immunoprecipitates from 293FT cells overexpressing Flag-NEDD4L and HA-ubiquitin that were incubated with 20 μM MG132 for 12 hr and pulled down by Flag or Flag and HA agarose. ( D) List of the top 3 candidate substrate proteins of NEDD4L identified via the mass spectrometry analysis. ( E) Representative western blots (left) and quantification of Myc-PRMT5 expression (normalized to β-actin expression, right) in control 293FT cells with HA-ubiquitin overexpression and 293FT cells with HA-ubiquitin overexpression and overexpression of wild-type NEDD4L (Flag-NEDD4L) or an E3 ligase activity-dead mutant of NEDD4L (Flag-NEDD4L C821A) transfected with or without Myc-PRMT5 and incubated with or without 20 μM MG132 for 12 hr. Three independent western blot analyses were performed. ( F) Representative western blots (left) and quantification of PRMT5 expression (normalized to β-actin expression, right) in control SW620-L1 cells (Control), and SW620-L1 cells overexpressing wild-type NEDD4L (NEDD4L) induced by treatment with 2 µg/ml doxycycline (for 24 hr) and incubated with or without 20 μM MG132 for 12 hr. Three independent western blot analyses were performed. ( G) Representative western blots (left) and quantification of PRMT5 expression (normalized to β-actin expression, right) in control HCT-15 cells (shControl), NEDD4L-knockdown HCT-15 cells (shNEDD4L) and HCT-15 cells with NEDD4L knockdown and restoration with wild-type NEDD4L resistant to shRNA targeting NEDD4L (shNEDD4L/NEDD4L-R) incubated with or without 20 μM MG132 for 12 hr. Three independent western blot analyses were performed. ( H) Representative western blots (left) and quantification of PRMT5 ubiquitination (normalized to PRMT5 expression, right) in control SW620-L1 cells (Control) and SW620-L1 cells with overexpression of wild-type NEDD4L (NEDD4L), an E3 ligase activity-dead mutant of NEDD4L (C821A) or a constitutively active mutant of NEDD4L (R776Q) induced by treatment with 2 µg/ml doxycycline (for 24 hr) and incubated with 20 μM MG132 for 12 hr. Three independent western blot analyses were performed. ( I) Representative western blots (left) and quantification of exogenous Flag-PRMT5 and a NEDD4L binding motif mutant of Flag-PRTM5 (normalized to β-actin expression, right) in control SW620-L1 cells and NEDD4L-overexpressing SW620-L1 cells with knockout of endogenous PRMT5 (sgPRMT5-1 or sgPRMT5-2) and overexpression of PRMT5 containing the wild-type NEDD4L binding motif (PPNAY), or mutant NEDD4L binding motif (PPNAA). To induce the overexpression of NEDD4L in these cells, the cells was induced by treatment with 2 µg/ml doxycycline (for 24 hr). Three independent western blot analyses were performed. The data are presented as the mean ± s.e.m. values. P -values were determined by unpaired two-way ANOVA with uncorrected Fisher’s LSD test (E, F, G, and I), or unpaired one-way ANOVA with uncorrected Fisher’s LSD test (H). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; n.s., not significant.

Article Snippet: To immunoprecipitate PRMT5, total lysates containing 1 mg of protein were incubated with 1 µg of a rabbit polyclonal anti-PRMT5 antibody overnight at 4°C and were then incubated with 20 µl of Protein A/G agarose (22851, Thermo Fisher Scientific) for 3 hours at 4°C.

Techniques: Staining, Immunoprecipitation, Over Expression, Incubation, Western Blot, Mass Spectrometry, Expressing, Control, Activity Assay, Mutagenesis, Transfection, Knockdown, shRNA, Binding Assay, Knock-Out

Journal: bioRxiv

Article Title: The E3 ligase NEDD4L prevents colorectal cancer liver metastasis via degradation of PRMT5 to inhibit the AKT/mTOR signaling pathway

doi: 10.1101/2024.10.21.619337

Figure Lengend Snippet: PRMT5 activates the AKT/mTOR signaling pathway to increase cell proliferation and promote colorectal cancer liver metastasis. ( A, B) Representative western blots (A) and quantification of p-mTOR levels (normalized to mTOR levels, B, left), p-AKT levels (normalized to AKT levels, B, middle), and p-4E-BP1 levels (normalized to 4E-BP1 levels, B, right) in control HCT-15 cells and HCT-15 cells with overexpression of WDR77 in combination with wild-type PRMT5 (PRMT5/WDR77) or a methyltransferase-inactive mutant of PRMT5 (PRMT5 R368A/WDR77) incubated with or without 200 μM amino acids (AA; for 15 min) and 800 nM insulin (for 10 min). Three independent western blot analyses were performed. ( C) In vitro proliferation assay of control HCT-15 cells (Control) and HCT-15 cells with overexpressing WDR77 in combination with wild-type PRMT5 (PRMT5/WDR77) or a methyltransferase-inactive mutant of PRMT5 (PRMT5 R368A/WDR77) (1,000 cells) cultured with 200 μM AA and 800 nM insulin for 24 hr. Five independent CCK-8 assays were performed. ( D, E) Representative western blots (D, left) and quantification of p-mTOR levels (normalized to mTOR levels, E, left), p-AKT levels (normalized to AKT levels, E, middle), and p-4E-BP1 levels (normalized to 4E-BP1 levels, E, right) in control SW620-L1 cells (sgControl) and PRMT5-knockout SW620-L1 cells (sgPRMT5-1 or sgPRMT5-2) incubated with or without 200 μM AA (for 15 min) and 800 nM insulin (for 10 min). Three independent western blot analyses were performed. ( F) In vitro proliferation assay of control SW620-L1 cells (sgControl) and PRMT5-knockout SW620-L1 cells (sgPRMT5-1 or sgPRMT5-2, 3,000 cells) cultured with 200 μM AA and 800 nM insulin for 24 hr. Five independent CCK-8 assays were performed. ( G) Representative western blots (left) and quantification of p-mTOR levels (normalized to mTOR levels, right) in control HCT-15 cells and HCT-15 cells overexpressing WDR77 in combination with wild-type PRMT5 (PRMT5/WDR77) cultured with or without 100 nM rapamycin (24 hr) in combination with 200 μM AA (for 15 min) and 800 nM insulin (for 10 min). Three independent western blot analyses were performed. ( H) In vitro proliferation assay of control HCT-15 cells (Control) and HCT-15 cells overexpressing WDR77 in combination with wild-type PRMT5 (PRMT5/WDR77) (1,000 cells) cultured with or without 100 nM rapamycin combined with 200 μM AA and 800 nM insulin for 24 hr. Five independent CCK-8 assays were performed. ( I) Bioluminescence imaging results (left) and quantification of liver metastases (right) in BALB/c nude mice implanted with control SW620-L1 cells (sgControl) or PRMT5-knockout SW620-L1 cells (sgPRMT5-1 or sgPRMT5-2, 1 × 10 6 cells) via intrasplenic injection. The n-values denote the number of mice per group. ( J) Bioluminescence imaging results (left) and quantification of liver metastases (right) in BALB/c nude mice implanted with control HCT-15 cells (Control), HCT-15 cells overexpressing WDR77 in combination with wild-type PRMT5 (PRMT5/WDR77) or HCT-15 cells overexpressing WDR77 in combination with a methyltransferase-inactive mutant of PRMT5 (PRMT5 R368A/WDR77) (3 × 10 6 cells) via intrasplenic injection. Rapamycin (2 mg/kg) was administered intraperitoneally every two days from the day cancer cell injection (day 0) to the experimental endpoint. The n-values denote the number of mice per group. The data are presented as the mean ± s.e.m. values. P -values were determined by unpaired two-way ANOVA with uncorrected Fisher’s LSD test (B, E, I, and J), or unpaired one-way ANOVA with uncorrected Fisher’s LSD test (C, F, G, and H). * P < 0.05; *** P < 0.001; **** P < 0.0001; n.s., not significant.

Article Snippet: To immunoprecipitate PRMT5, total lysates containing 1 mg of protein were incubated with 1 µg of a rabbit polyclonal anti-PRMT5 antibody overnight at 4°C and were then incubated with 20 µl of Protein A/G agarose (22851, Thermo Fisher Scientific) for 3 hours at 4°C.

Techniques: Western Blot, Control, Over Expression, Mutagenesis, Incubation, In Vitro, Proliferation Assay, Cell Culture, CCK-8 Assay, Knock-Out, Imaging, Injection

Journal: bioRxiv

Article Title: The E3 ligase NEDD4L prevents colorectal cancer liver metastasis via degradation of PRMT5 to inhibit the AKT/mTOR signaling pathway

doi: 10.1101/2024.10.21.619337

Figure Lengend Snippet: PRMT5 methylates AKT1 R391 to activate the AKT/mTOR signaling pathway and increase colorectal cancer cell proliferation. ( A) Representative western blots of Flag-PRMT5 immunoprecipitates and total lysates of control 293FT cells and 293FT cells with HA-AKT, HA-TSC2, or Myc-Rheb overexpression with or without Flag-PRMT5 and Myc-WDR77 overexpression. Three independent western blot analyses were performed. ( B) Representative western blots showing PRMT5 immunoprecipitates and total lysates of SW620-L1 cells incubated with 200 μM amino acids (AA; for 15 min) and 800 nM insulin (for 10 min). Three independent western blot analyses were performed. ( C) Representative western blots (left) and quantification of AKT1 methylarginine levels (normalized to AKT1 expression, right) in control SW620-L1 cells (sgControl) and PRMT5-knockout SW620-L1 cells (sgPRMT5-1 or sgPRMT5-2) incubated with 200 μM AA (for 15 min) and 800 nM insulin (for 10 min). Three independent western blot analyses were performed. ( D) Representative western blots (left) and quantification of AKT1 methylarginine levels (normalized to AKT1 levels, right) in SW620-L1 cells treated with EPZ015666 (0, 0.31 or 1.25 μM, for 48 h) and incubated with 200 μM AA (for 15 min) and 800 nM insulin (for 10 min). Three independent western blot analyses were performed. ( E, F) Representative western blots (E) and quantification of p-mTOR levels (normalized to mTOR levels, F, left), p-AKT levels (normalized to AKT levels, F, middle), and p-4E-BP1 levels (normalized to 4E-BP1 levels, F, right) in HCT-15 cells with PRMT5 overexpression in combination with double knockout of endogenous AKT1 and AKT2 and overexpression of wild-type AKT1 (PRMT5/sgAKT1-1/sgAKT2/AKT1 or PRMT5/sgAKT1-2/sgAKT2/AKT1), or the AKT1 R391K mutant that cannot be methylated by PRMT5 (PRMT5/sgAKT1-1/sgAKT2/AKT1-R391K or PRMT5/sgAKT1-2/sgAKT2/AKT1-R391K) incubated with 200 μM AA (for 15 min) and 800 nM insulin (for 10 min). Three independent western blot analyses were performed. ( G) In vitro proliferation assay of HCT-15 cells with overexpression of PRMT5 and WDR77 in combination with double knockout of endogenous AKT1 and AKT2 and overexpression of wild-type AKT1 (PRMT5/WDR77/sgAKT1-1/sgAKT2/AKT1 or PRMT5/WDR77/sgAKT1-2/sgAKT2/AKT1) or the AKT1-R391K mutant that cannot be methylated by PRMT5 (PRMT5/WDR77/sgAKT1-1/sgAKT2/AKT1 R391K or PRMT5/WDR77/sgAKT1-2/sgAKT2/AKT1 R391K) (1,000 cells) cultured with 200 μM AA and 800 nM insulin for 24 hr. Five independent CCK-8 assays were performed. The data are presented as the mean ± s.e.m. values. P -values were determined by unpaired one-way ANOVA with uncorrected Fisher’s LSD test (C and D), or unpaired two-way ANOVA with uncorrected Fisher’s LSD test (F and G). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: To immunoprecipitate PRMT5, total lysates containing 1 mg of protein were incubated with 1 µg of a rabbit polyclonal anti-PRMT5 antibody overnight at 4°C and were then incubated with 20 µl of Protein A/G agarose (22851, Thermo Fisher Scientific) for 3 hours at 4°C.

Techniques: Western Blot, Control, Over Expression, Incubation, Expressing, Knock-Out, Double Knockout, Mutagenesis, Methylation, In Vitro, Proliferation Assay, Cell Culture, CCK-8 Assay

Journal: bioRxiv

Article Title: The E3 ligase NEDD4L prevents colorectal cancer liver metastasis via degradation of PRMT5 to inhibit the AKT/mTOR signaling pathway

doi: 10.1101/2024.10.21.619337

Figure Lengend Snippet: NEDD4L promotes PRMT5 degradation to inhibit the AKT/mTOR signaling pathway and prevent colorectal cancer liver metastasis. ( A) Representative western blots (left) and quantification of AKT1 methylarginine levels (normalized to AKT1 levels, right) in control HCT-15 cells (shControl), NEDD4L-knockdown HCT-15 cells (shNEDD4L) and HCT-15 cells with knockdown of NEDD4L and restoration with wild-type NEDD4L resistant to shRNA targeting NEDD4L (shNEDD4L/NEDD4L-R) incubated with 200 μM amino acid (AA; for 15 min) and 800 nM insulin (for 10 min). Three independent western blot analyses were performed. ( B) Representative western blots (left) and quantification of AKT1 methylarginine levels (normalized to AKT1 expression, right) in control SW620-L1 cells (Control) and SW620-L1 cells overexpressing wild-type NEDD4L (NEDD4L) or an E3 ligase activity-dead mutant of NEDD4L (C821A) induced by treatment with 2 µg/ml doxycycline (for 24 hr) and incubated with 200 μM AA (for 15 min) and 800 nM insulin (for 10 min). Three independent western blot analyses were performed. ( C, D) Representative western blots (C) and quantification of p-mTOR levels (normalized to mTOR levels, D, left), p-AKT levels (normalized to AKT levels, D, middle), and p-4E-BP1 levels (normalized to 4E-BP1 levels, D, right) in control HCT-15 cells (shControl/sgControl), HCT-15 cells with NEDD4L knockdown (shNEDD4L/sgControl), and HCT-15 cells with NEDD4L knockdown in combination with knockout of PRMT5 (shNEDD4L/sgPRMT5-1 or shNEDD4L/sgPRMT5-2) incubated with or without 200 μM AA (for 15 min) and 800 nM insulin (for 10 min). Three independent western blot analyses were performed. ( E) In vitro proliferation assay of control HCT-15 cells (shControl/sgControl), HCT-15 cells with knockdown of NEDD4L (shNEDD4L/sgControl), and HCT-15 cells with knockdown of NEDD4L in combination with knockout of PRMT5 (shNEDD4L/sgPRMT5-1 or shNEDD4L/sgPRMT5-2) (1,000 cells) cultured with 200 μM AA and 800 nM insulin for 24 hr. Five independent CCK-8 assays were performed. ( F-H) Representative western blots (F) and quantification of p-mTOR levels (normalized to mTOR levels, G, left), p-AKT levels (normalized to AKT levels, G, right), and p-4E-BP1 levels (normalized to 4E-BP1 levels, H) in SW620-L1 cells with overexpression of NEDD4L and WDR77 (NEDD4L-WDR77) in combination with knockout of endogenous PRMT5 (sgPRMT5-1 or sgPRMT5-2) and overexpression of PRMT5 containing the wild-type NEDD4L binding motif (PPNAY) or mutant NEDD4L binding motif (PPNAA) incubated with or without 200 μM AA (for 15 min) and 800 nM insulin (for 10 min). NEDD4L overexpression in cancer cells was induced by treatment with 2 µg/ml doxycycline (for 24 hr). Three independent western blot analyses were performed. ( I) In vitro proliferation assay of SW620-L1 cells with overexpression of NEDD4L and WDR77 (NEDD4L-WDR77) in combination with knockout of endogenous PRMT5 (sgPRMT5-1 or sgPRMT5-2) and overexpression of PRMT5 containing wild-type NEDD4L binding motif (PPNAY) or mutant NEDD4L binding motif (PPNAA) (3,000 cells) cultured with 200 μM AA and 800 nM insulin for 24 hr. Five independent CCK-8 assays were performed. ( J) Bioluminescence imaging results(left) and quantification of liver metastases (right) in BALB/c nude mice implanted with control HCT-15 cells (shControl/sgControl), HCT-15 cells with knockdown of NEDD4L (shNEDD4L/sgControl), and HCT-15 cells with knockdown of NEDD4L in combination with knockout of PRMT5 (shNEDD4L/sgPRMT5-1 or shNEDD4L/sgPRMT5-2) (3 × 10 6 cells) via intrasplenic injection. The n-values denote the number of mice per group. The data are presented as the mean ± s.e.m. values. P -values were determined by unpaired one-way ANOVA with uncorrected Fisher’s LSD test (A, B, and E), or unpaired two-way ANOVA with uncorrected Fisher’s LSD test (D, G, H, I, and J). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; n.s., not significant.

Article Snippet: To immunoprecipitate PRMT5, total lysates containing 1 mg of protein were incubated with 1 µg of a rabbit polyclonal anti-PRMT5 antibody overnight at 4°C and were then incubated with 20 µl of Protein A/G agarose (22851, Thermo Fisher Scientific) for 3 hours at 4°C.

Techniques: Western Blot, Control, Knockdown, shRNA, Incubation, Expressing, Activity Assay, Mutagenesis, Knock-Out, In Vitro, Proliferation Assay, Cell Culture, CCK-8 Assay, Over Expression, Binding Assay, Imaging, Injection

Journal: bioRxiv

Article Title: The E3 ligase NEDD4L prevents colorectal cancer liver metastasis via degradation of PRMT5 to inhibit the AKT/mTOR signaling pathway

doi: 10.1101/2024.10.21.619337

Figure Lengend Snippet: The mechanism by which NEDD4L prevents colorectal cancer liver metastasis. In colorectal cancer cells with high expression of NEDD4L, E3 ligase NEDD4L binds to PPNAY motif in PRMT5 resulting in ubiquitination and degradation of PRMT5. PRMT5 degradation attenuates the methylation of an arginine residue in AKT1 to inhibit AKT/mTOR signaling pathway, consequently decreasing colorectal cancer cell proliferation and ultimately preventing colorectal cancer liver metastasis. Conversely, in colorectal cancer cells with low expression of NEDD4L, the level of NEDD4L is not sufficient for ubiquitination and degradation of PRMT5, resulting in an increase in AKT1 methylarginine to activate the AKT/mTOR signaling pathway. The activation of the AKT/mTOR signaling pathway increases colorectal cancer cell proliferation and ultimately promotes colorectal cancer liver metastasis.

Article Snippet: To immunoprecipitate PRMT5, total lysates containing 1 mg of protein were incubated with 1 µg of a rabbit polyclonal anti-PRMT5 antibody overnight at 4°C and were then incubated with 20 µl of Protein A/G agarose (22851, Thermo Fisher Scientific) for 3 hours at 4°C.

Techniques: Expressing, Methylation, Residue, Activation Assay

Journal: iScience

Article Title: The PRMT5-splicing axis is a critical oncogenic vulnerability that regulates detained intron splicing

doi: 10.1016/j.isci.2025.112965

Figure Lengend Snippet: PRMT5 inhibition causes the formation of DIs specifically in the nucleus (A–C) U87 cells were treated with vehicle or 10 nM JNJ-64619178 (PRMT5i) for 3 days and polyA + mRNA was deep sequenced and analyzed to identify and quantify exon-based alternative splicing (AS) events, including DIs, as shown in the schematic (A). (B) Dots represent individual events for each AS type, with color denoting those significantly (p adj < 0.05) or not significantly (gray) altered by treatment. (C) Proportion of each AS event type that is significantly (p adj < 0.05) altered by treatment. The total number of significant AS events is indicated above each bar. (D–F) U87 cells were treated with vehicle or PRMT5i (as above). Nuclear and cytoplasmic fractions were isolated prior to sequencing (as above). (D) Heatmap shows intron-containing isoforms that are significantly up- or down-regulated by PRMT5i-treatment in either nuclear or cytoplasmic compartments (p adj < 0.05). Columns show replicates, rows indicate individual introns, and the coloring indicates Z score normalized isoform counts where red denotes higher expression and blue denotes lower expression. (E) For all PRMT5-regulated DI isoforms ( Left ), and their coding isoforms ( Right ). The y axis values show the proportion of reads present in nuclear versus cytoplasmic fractions (1 – exclusively nuclear, 0 – equal distribution, −1 – exclusively cytoplasmic). The x axis values show the proportion of reads in vehicle versus PRMT5i-treated samples (1 – exclusively in PRMT5i samples, 0 – equal distribution, −1 – exclusively in vehicle samples). Each dot represents a specific isoform. (F) Representative sequencing tracks for a DI (red), and its adjacent exons (gray), in DNA2 in nuclear or cytoplasmic fractions and in whole cell lysates, with or without PRMT5i treatment. (G) Comparison of the induction of PRMT5-regulated DIs ( Left ) and their coding isoforms ( Right ) detected in nuclear (DIs) or cytoplasmic (coding) fractions versus whole cell lysate. Inset shows the region displayed in the larger graphs. Correlation values reflect the Pearson correlation coefficient. (H) Bar graphs ( Top ) show the quantification of DI (red) and coding (gray) isoform levels for six representative PRMT5-resposive genes (as indicated), determined by qRT-PCR of total RNA from whole cell lysates of U87 cells treated with vehicle or 10 nM JNJ-64619178 (PRMT5i) for 3 days. Data are mean ± SD of 3 technical replicates. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001, Student’s t test. Protein levels ( Bottom ) were determined by western, using GAPDH as a loading control. Quantifications are shown below each blot. For this and all subsequent western blots, numbers indicate approximate molecular weight in kilodaltons.

Article Snippet: Rabbit anti-PRMT5 Polyclonal Antibody , Cell Signaling Technology , Cat#2252; RRID: AB_10694541.

Techniques: Inhibition, Alternative Splicing, Isolation, Sequencing, Expressing, Comparison, Quantitative RT-PCR, Western Blot, Control, Molecular Weight

Journal: iScience

Article Title: The PRMT5-splicing axis is a critical oncogenic vulnerability that regulates detained intron splicing

doi: 10.1016/j.isci.2025.112965

Figure Lengend Snippet: PRMT5 regulates a conserved set of DIs across multiple human cell lines (A) Human cell lines used for AS analysis, including cancer or tissue of origin, driver mutations or construct, and MTAP status. (B) Proportion of each AS event type that show significantly altered levels in the indicated cell line in response to 3-day treatment with 10 nM JNJ-64619178 (PRMT5i) compared to vehicle control (p adj < 0.05). The total number of significant AS events for that cell line is indicated to the right of each bar. (C) Relative expression of individual DIs across all human cell lines after 3-day vehicle or PRMT5i treatment. Each column represents a specific DI that is significantly up- or down-regulated by PRMT5i-treatment in at least one cell line. Coloring represents Z score normalized intron counts, where red indicates higher relative expression and blue indicates lower relative expression. (D) Similarity matrix of the indicated AS event significantly altered by PRMT5i treatment (p adj < 0.05). Each square represents the similarity between the two intersecting cell lines, with coloring indicating percent similarity (dice similarity score) and square size indicating the number of similar events. (E) Bar charts of DI conservation across 7 human cell lines. Bar length represents the number of significant PRMT5i-upregulated DIs (p adj < 0.05, log 2 FC > 0) for each cell line and the color denotes the number of cell lines in which a given DI is conserved across. The total number of significant DIs in each line is indicated to the right of each bar. (F) Enriched GO terms from the KW Biological Process gene set for PRMT5i-upregulated DIs in each cell line (p adj < 0.05, log 2 FC > 0). Terms are displayed if they are significant in at least one cell line (FDR < 0.01). Color represents enrichment score, while size inversely correlates with significance value.

Article Snippet: Rabbit anti-PRMT5 Polyclonal Antibody , Cell Signaling Technology , Cat#2252; RRID: AB_10694541.

Techniques: Construct, Control, Expressing

Journal: iScience

Article Title: The PRMT5-splicing axis is a critical oncogenic vulnerability that regulates detained intron splicing

doi: 10.1016/j.isci.2025.112965

Figure Lengend Snippet: Generation of endogenous dTag-CLNS1A in multiple human cell lines to specifically inhibit the PRMT5-splicing axis (A) Schematic indicating how use of the dTag-CLNS1A degron system specifically separates the disruption of the methylosome from other PRMT5 functions. (B) Western blot analysis of representative dTag-CLNS1A clones for CAL51, HEK293T, and HCT116 cells, alongside parental controls with antibodies against CLNS1A, the inserted HA tag, or loading control GAPDH. Asterisk (∗) marks a non-specific band. (C) Dose-response curves for three parental cell lines treated with dTag-13 for 6 days. Data are mean ± SD of 3 technical replicates/line. Indicated p -values represent the most significant comparison between the respective drug concentration and vehicle-treated cells. Vertical dashed line represents the concentration above which dTag-13 has off-target effects. ∗∗∗∗ p < 0.0001, Student’s t test. (D) Western blot analyses of HA (CLNS1A) and loading control GAPDH in four CAL51, HEK293T, and HCT116 dTag-CLNS1A clones treated with either vehicle or 1 μM dTag-13 for 3 days.

Article Snippet: Rabbit anti-PRMT5 Polyclonal Antibody , Cell Signaling Technology , Cat#2252; RRID: AB_10694541.

Techniques: Disruption, Western Blot, Clone Assay, Control, Comparison, Concentration Assay

Journal: iScience

Article Title: The PRMT5-splicing axis is a critical oncogenic vulnerability that regulates detained intron splicing

doi: 10.1016/j.isci.2025.112965

Figure Lengend Snippet: HCT116 cells are CLNS1A-independent until PRMT5 activity is decreased (A) Western blot analyses of HA (CLNSIA), SDMA (methyl-SmB), and HSP90 as loading control in HCT116 dTag-CLNS1A #1 ( Left ) and #2 ( Right ) maintained in 1 μM dTag-13 for the indicated number of days. (B) Representative Western blot analysis of CLNS1A, SDMA (methyl-SmB), and loading control GAPDH levels in HCT116 sgCtrl and four sgCLNS1A clones ( n = 4 biological replicates). (C) Relative proliferation of HCT116 sgCtrl and four sgCLNS1A clones through analyses of cumulative population doubling over a 6-day period. Data represent mean ± SD of 10 technical replicates. (D) Relative viability of HCT116 sgCtrl and indicated sgCLNS1A clones treated with the PRMT5i JNJ-64619178 for 6 days. Data represent mean ± SD of 3 technical replicates. (E–G) HCT116 parental or two dTag-CLNS1A clones treated with or without 1μM dTag-13 in combination with the indicated concentrations of JNJ-64619178 (PRMT5i). (E) Relative viability was assessed after treatment for 6 days. Data represent mean ± SD of 3 technical replicates. Red markings correspond to the critical PRMT5i doses used in F and 5G. (F) Western blot analyses of HA (CLNS1A), SDMA, and loading control HSP90 after treatment with the critical indicated PRMT5i doses for 3 days (G) Relative levels of a representative DI in EIF4E were assessed by qRT-PCR after treatment with the critical indicated PRMT5i doses for 3 days. Data represent mean ± SD of 3 technical replicates. For all panels, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001, Student’s t test.

Article Snippet: Rabbit anti-PRMT5 Polyclonal Antibody , Cell Signaling Technology , Cat#2252; RRID: AB_10694541.

Techniques: Activity Assay, Western Blot, Control, Clone Assay, Quantitative RT-PCR

Journal: iScience

Article Title: The PRMT5-splicing axis is a critical oncogenic vulnerability that regulates detained intron splicing

doi: 10.1016/j.isci.2025.112965

Figure Lengend Snippet: PRMT5 regulates a conserved set of DIs in multiple mouse cell lines (A) Summary of mouse cell lines used for AS analysis, including cancer type, driver mutations, PRMT5i used, and MTAP status. (B) Proportion of each AS event type that is significantly different in indicated cell line after 3-day PRMT5i treatment compared to vehicle control (p adj < 0.05). Total number of significant AS events is indicated above the bar. (C) Relative expression of individual DIs across all mouse cell lines after 3-day vehicle or PRMT5i treatment. Each column represents a specific DI that is significantly upregulated or downregulated by PRMT5i-treatment in at least one cell line. Colors represent column Z score normalized intron counts, where red indicates higher relative expression and blue indicates lower relative expression. (D) Similarity matrix of the indicated class of AS event that was significantly altered by PRMT5i treatment. Each square represents the similarity between the two intersecting murine cell lines. Square coloring indicates percent similarity (dice similarity score), and square size is proportional to the number of similar events. (E) Bar charts of DI conservation across all mouse cell lines. Each bar represents the number of PRMT5i-upregulated DIs (p adj < 0.05, log 2 FC > 0) in each cell line and the color corresponds to the number of cell lines in which a given DI is conserved across. Total number of significant DIs in each line is indicated to the right of the bar. (F) Enriched GO terms from the KW Biological Process gene set for PRMT5i-upregualted DIs in each cell line (p adj < 0.05, log 2 FC > 0). Terms are displayed if they are significant in at least one cell line (FDR < 0.01). Color represents enrichment score, while size inversely correlates with significance value.

Article Snippet: Rabbit anti-PRMT5 Polyclonal Antibody , Cell Signaling Technology , Cat#2252; RRID: AB_10694541.

Techniques: Control, Expressing

Journal: iScience

Article Title: The PRMT5-splicing axis is a critical oncogenic vulnerability that regulates detained intron splicing

doi: 10.1016/j.isci.2025.112965

Figure Lengend Snippet: PRMT5-regulated DI identity is not conserved across species, but it occurs in genes in similar complexes to regulate proliferation-associated processes (A) Overlap of common DI-regulated ( Left ) processes, ( Center ) genes, and ( Right ) complexes for PRMT5-regulated DIs in human (H23) and mouse (KP393T5) lung cancer cell lines. Percentages represent the dice similarity score for each comparison. (B) Enrichment of DI-regulated biological processes in human and mouse cells. Each pair of bars represents a specific biological process, with the blue bar representing the FDR of the human H23 lung cancer line and the red bar representing the FDR of the mouse KP393T5 lung cancer line. Vertical dashed line represents the significance cutoff (FDR < 0.05). (C) Schematic of representative DI-regulated complexes, including the minichromosome maintenance (MCM) and mediator (MED) complexes. Each shape represents a specific complex member, and a color fill of blue, red, purple, or white indicates that the complex member contained a DI in human, mouse, both species, or neither, respectively. (D) Sankey plot showing convergence of DI-regulated complexes and biological processes. Each line represents a specific DI moving from ( Column 1 ) gene to ( Column 2 ) complexes to ( Column 3 ) biological processes. Line color signifies conservation at the ( Left ) gene or ( Right ) complex level, and thickness corresponds to the number of ( Left ) complexes or ( Right ) biological processes a given gene is a part of. Column color represents conservation at the level of ( Column 1 ) gene, ( Column 2 ) complexes, and ( Column 3 ) biological processes. Blue indicates unique to human cells, red unique to mouse cells, and purple common to both species. For biological processes, white indicates DIs/complexes that map to non-significant biological processes (FDR > 0.05).

Article Snippet: Rabbit anti-PRMT5 Polyclonal Antibody , Cell Signaling Technology , Cat#2252; RRID: AB_10694541.

Techniques: Comparison

Journal: Cell reports

Article Title: PRMT5 mediates FoxO1 methylation and subcellular localization to regulate lipophagy in myogenic progenitors

doi: 10.1016/j.celrep.2023.113329

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal anti-PRMT5 , Proteintech , Cat# 18436–1-AP, RRID: AB_2171798.

Techniques: Recombinant, Red Blood Cell Lysis, Modification, Saline, Blocking Assay, Plasmid Preparation, Reverse Transcription, Bicinchoninic Acid Protein Assay, Western Blot, In Situ, Software

Journal: bioRxiv

Article Title: Protein arginine methyltransferase 5 (Prmt5) localizes to chromatin loop anchors and modulates expression of genes at TAD boundaries during early adipogenesis

doi: 10.1101/2023.06.13.544859

Figure Lengend Snippet: Genome-wide binding patterns for Prmt5. (A) Genome Browser tracks showing Prmt5 ChIP-seq at day 0, day 1, and day 2 of 3T3-L1 differentiation. Significant ChIP peaks determined by MACS2 are indicated below the genome browser tracks in gray for the Abhd2 promoter and (B) the Pdgfra promoter. (C) Gene annotation of all significant Prmt5 peaks at day 0. (D) Top biological processes identified from Gene Ontology analysis of significant day 0 Prmt5 peaks. (E) The HOMER motif discovery algorithm identified Runx1 and Nrf1 as the top motifs present within Prmt5 peaks. (F) Prmt5 ChIP-Seq tag density plots over the gene body ± 2 kb displayed as heat maps, order determined by k-means clustering. Expression data displayed as log 2 TPM (transcripts per million mapped reads) for day 0, day 1, day 2, and day 7 of 3T3-L1 differentiation.

Article Snippet: For chromatin immunoprecipitation (ChIP) experiments, rabbit polyclonal Prmt5 antibody (Epigentek, catalog # A-3005-100) and normal Rabbit IgG (Cell Signaling Technology, catalog # 2729) were used.

Techniques: Genome Wide, Binding Assay, ChIP-sequencing, Expressing

Journal: bioRxiv

Article Title: Protein arginine methyltransferase 5 (Prmt5) localizes to chromatin loop anchors and modulates expression of genes at TAD boundaries during early adipogenesis

doi: 10.1101/2023.06.13.544859

Figure Lengend Snippet: Association between histone modifications and Prmt5 binding. (A) Average tag density plots for Prmt5, H3K27ac, H3K4me3, and H3K27me3 ChIP-Seq, in relation to Prmt5 peak centers. (B) Tag density plots for day 0 and day 2 ChIP-Seq for Prmt5, H3K27ac, H3K4me3, and H3K27me3, plotted +/- 2kb of Prmt5 peak centers. (C) Genome Browser tracks showing Prmt5 ChIP-seq at day 0, day 1, and day 2 of 3T3-L1 differentiation, along with the corresponding tracks for H3K27ac, H3K4me3, H3K27me3 ChIP-Seq at day 0 and day 2 of 3T3-L1 differentiation. Significant ChIP peaks determined by MACS2 are indicated below the genome browser tracks. (D) bedtools intersect was used to calculate significant peak overlap between Prmt5 ChIP-Seq and histone modification ChIP-Seqs at day 0 and day 2 of differentiation.

Article Snippet: For chromatin immunoprecipitation (ChIP) experiments, rabbit polyclonal Prmt5 antibody (Epigentek, catalog # A-3005-100) and normal Rabbit IgG (Cell Signaling Technology, catalog # 2729) were used.

Techniques: Binding Assay, ChIP-sequencing, Modification

Journal: bioRxiv

Article Title: Protein arginine methyltransferase 5 (Prmt5) localizes to chromatin loop anchors and modulates expression of genes at TAD boundaries during early adipogenesis

doi: 10.1101/2023.06.13.544859

Figure Lengend Snippet: Prmt5-dependent gene expression changes. (A) Genome Browser tracks showing Prmt5 ChIP-seq at day 0, day 1, and day 2 of 3T3-L1 differentiation. Significant ChIP peaks determined by MACS2 are immediately below the genome browser tracks in gray. (B) Real- time RT-qPCR analysis of Prmt5-bound genes in 3T3-L1 cells transfected with scrambled or Prmt5 -targeting siRNA. Cells were collected at time points between D0 and D4 of differentiation. Data represent averages from 3-6 replicates and are presented as means +/- standard deviations (SD). The level of expression in scrambled day 0 samples were set to a value of 1 for genes Ptn , Thbs2 , and Pdgfra , and other values are relative to that sample value. *, P < 0.05; **, P < 0.001; ***, P < 0.0001 (versus scrambled by Student’s t test). (C) Volcano plots displaying differentially expressed genes between scrambled and Prmt5 siRNA transfected 3T3-L1 cells harvested at day 0 of differentiation. The y-axis data correspond to the mean log10 expression levels (padjusted-values). The red dots represent significantly upregulated genes (padj < 0.05, log 2 foldchange > 0.5) and blue dots represent significantly downregulated genes (padj < 0.05, log 2 foldchange < −0.5). The black dots represent genes whose differential expression levels did not reach statistical significance. Flanking the volcano plot are Gene Ontology Biological processes charts for the top upregulated and downregulated genes. (D) Differential expression of Prmt5-bound Prmt5- dependent genes ranked by log 2 foldchange displayed on the right, with corresponding day 0 Prmt5 ChIP-Seq tag density plots over the gene body ± 2 kb displayed to the left.

Article Snippet: For chromatin immunoprecipitation (ChIP) experiments, rabbit polyclonal Prmt5 antibody (Epigentek, catalog # A-3005-100) and normal Rabbit IgG (Cell Signaling Technology, catalog # 2729) were used.

Techniques: Gene Expression, ChIP-sequencing, Quantitative RT-PCR, Transfection, Expressing, Quantitative Proteomics

Journal: bioRxiv

Article Title: Protein arginine methyltransferase 5 (Prmt5) localizes to chromatin loop anchors and modulates expression of genes at TAD boundaries during early adipogenesis

doi: 10.1101/2023.06.13.544859

Figure Lengend Snippet: Co-localization of Prmt5 with loop anchors and regulators of genome structure. (A) Genome Browser tracks for showing Prmt5 ChIP-seq at day 0 of 3T3-L1 differentiation, along with the corresponding tracks from H3K27ac, H3K4me3, Med1, Smc1, and Ctcf ChIP- Seqs with D0 Promoter Capture Hi-C displayed above. Significant ChIP peaks determined by MACS2 are indicated below the genome browser tracks. (B) Average tag density plots for Prmt5, Ctcf, Med1, and Smc1 ChIP-Seq, in relation to Prmt5 peak centers. (C) Tag density plots for day 0 and day 2 ChIP-Seq for Prmt5, Med1, Smc1, and Ctcf, plotted +/- 2kb of Prmt5 peak centers. (D) bedtools intersect was used to calculate significant peak overlap between Prmt5 ChIP-Seq and Med1, Smc1, and Ctcf ChIP-Seqs at day 0 and day 2 of differentiation. (E) Average tag density plots showing Smc1, Med1 and Prmt5 binding at DNA loop anchors +/-2kb, in relation to Ctcf binding.

Article Snippet: For chromatin immunoprecipitation (ChIP) experiments, rabbit polyclonal Prmt5 antibody (Epigentek, catalog # A-3005-100) and normal Rabbit IgG (Cell Signaling Technology, catalog # 2729) were used.

Techniques: ChIP-sequencing, Hi-C, Binding Assay

Journal: bioRxiv

Article Title: Protein arginine methyltransferase 5 (Prmt5) localizes to chromatin loop anchors and modulates expression of genes at TAD boundaries during early adipogenesis

doi: 10.1101/2023.06.13.544859

Figure Lengend Snippet: Effect of Prmt5 knockdown on TADs and TAD boundaries. (A) Compartment profiles (the first principal components) of scrambled siRNA and Prmt5 siRNA data for Chr 1. The A-type (open) compartments are shown in green, and the B-type (closed) compartments are shown in red. Below this, contact heatmaps are displayed. (B) Insulation plot profiles at 10-kb intervals are displayed genomic loci around genes Cdk14 and Myc for scrambled siRNA and Prmt5 siRNA transfected samples. Below that, a genome browser track for the same locus is displayed showing Prmt5 and Ctcf ChIP-seq peaks at day 0 of 3T3-L1 differentiation. High-confidence D0 3T3-L1 looping event interactions are depicted as purple lines below this plot. (C) Insulation scores were plotted for all Prmt5-bound TAD boundaries for day 0 scrambled siRNA and Prmt5 siRNA transfected samples.

Article Snippet: For chromatin immunoprecipitation (ChIP) experiments, rabbit polyclonal Prmt5 antibody (Epigentek, catalog # A-3005-100) and normal Rabbit IgG (Cell Signaling Technology, catalog # 2729) were used.

Techniques: Knockdown, Insulation, Transfection, ChIP-sequencing

Journal: eLife

Article Title: Type I and II PRMTs inversely regulate post-transcriptional intron detention through Sm and CHTOP methylation

doi: 10.7554/eLife.72867

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-human PRMT5 (Rabbit polyclonal) , Millipore , Cat#: 07-405RRID: AB_310589 , WB: 1:2000.

Techniques: Transfection, Construct, Mutagenesis, Plasmid Preparation, Software

Journal: eLife

Article Title: PRMT5 regulates ovarian follicle development by facilitating Wt1 translation

doi: 10.7554/eLife.68930

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-PRMT5 (rabbit polyclonal) , Millipore , Cat# 07-405 , IF (1:200),WB (1:1000).

Techniques: Recombinant, Plasmid Preparation, Luciferase, Reporter Assay