Journal: Science Advances

Article Title: FeaSion decodes the regulatory landscape and functional diversity of RNA polymerase II CTD phosphorylation

doi: 10.1126/sciadv.adz2345

Figure Lengend Snippet: ( A ) Stacked bar plot showing the kinases (left) and phosphatases (right) identified in RNAPII ChIP-MS, CRISPR screening and RNAPII mut ChIP-MS. ( B ) Pie plots showing the family of kinases identified in CRISPR screening. ( C ) Heatmap showing the NormZ of CRISPR screening for CLK1, CLK4, and YES1. ( D ) Western blot analyses of the RNAPII, pY1, pS2, pT4, pS5, and pS7 in CLK1, CLK4, or YES1 KO cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control. ( E ) Metagene profiles and boxplots (top) and log 2 (fold change) metagene heatmaps (bottom) showing pS7 and RNAPII ChIP-seq density in control, CLK1 KO, and CLK4 KO cells, as well as in untreated and TG003-treated (20 μM, 4 hours) cells (top). Statistical significance was assessed by a two-sided Wilcoxon test. *** P < 0.001. The value on the box plot indicating the fold change. ( F ) Similar to (E), but for pY1 and RNAPII ChIP-seq in control and YES1 KO cells, as well as in untreated and CHR6953755-treated (10 μM, 24 hours) cells. ( G ) Schematic workflow of in vitro phosphorylation assay. ( H ) In vitro phosphorylation activity of CLK1 (left), CLK4 (middle), and YES1 (right) on CTD. The CTD was detected using HA tag, and CLK1, CLK4, and YES1 were detected using Flag tag. ( I ) Pie chart (top) and metagene heatmap (bottom) showing the distribution of the top 500 CLK1 (left) CLK4 (middle) and YES1 (right) ChIP-seq binding sites and their densities. ( J ) Bar plot showing the gene ontology enrichment result of CLK1 (top), CLK4 (middle) and YES1 (bottom) binding genes. ( K ) Tracks of ING5 of CLK1 (left), DLX2 of CLK4 (middle), and IMPACT of YES1 (right) ChIP-seq signals and the changes of pS7 or pY1 and RNAPII after their knockout. miRNA, microRNA; snoRNA, small nucleolar RNA. TES, transcription end site.

Article Snippet: The membrane was blocked with 5% nonfat dry milk at room temperature for 30 min and then incubated overnight at 4°C with the RPB1 NTD antibody (Cell Signaling Technology, #14958), phospho-RPB1 CTD (Ser 2 ) antibody (Cell Signaling Technology, #13499), phospho-RPB1 CTD (Ser 5 ) antibody (Cell Signaling Technology, #13523), phospho-RPB1 CTD (Tyr 1 ) antibody (Merck Millipore, #MABE350), phospho-RPB1 CTD (Thr 4 ) antibody (Cell Signaling Technology, #26319), phospho-RPB1 CTD (Ser 7 ) antibody (Cell Signaling Technology, #13780), CLK1 antibody (Santa Cruz Biotechnology, #sc-515897), CLK4 antibody (Proteintech, #31205-1-AP), YES1 antibody (Proteintech, #20243-1-AP), α-tubulin antibody (Proteintech, #66031-1-Ig), GFP antibody (Proteintech, #50430-2-AP), HA antibody (Proteintech, #51064-2-AP), or glyceraldehyde-3-phosphate dehydrogenase antibody (Proteintech, #10494-1-AP) to the target protein.

Techniques: CRISPR, Western Blot, Control, ChIP-sequencing, In Vitro, Phospho-proteomics, Activity Assay, FLAG-tag, Binding Assay, Knock-Out

Journal: bioRxiv

Article Title: Dephosphorylation of YES kinase-mediated co-chaperone DNAJB6b phosphorylation attenuates tau protein aggregation

doi: 10.1101/2025.11.14.688390

Figure Lengend Snippet: (A) The phosphorylation of DNAJB6b at the Y53 site was evaluated in NVP-AEW541-treated SH-SY5Y cells by western blotting. Cells were over-expressed with empty vector (pcDNA/FRT/TO-V5) and indicated V5-DNAJB6b constructs for 48 hours, followed by NVP-AEW541 treatment or solvent control DMSO for 2 hours. Proteins were analyzed by immunoblotting using the indicated antibodies. (B-C) Quantification of the ratio of p-DNAJB6b Y53 to DNAJB6b wild-type or DNAJB6b Y53F (B) and the ratio of p-AKT Ser473 to AKT (C) in (A) ( n =3 ) . (D) The phosphorylation level of DNAJB6b Y53 was evaluated in dasatinib-treated cells by western blotting. SH-SY5Y cells were overexpressed with empty vector (pcDNA/FRT/TO-V5) and indicated V5-DNAJB6b constructs for 48 hours, followed by dasatinib treatment or DMSO solvent control for 2 hours. Proteins were analyzed by immunoblotting using the indicated antibodies. (E-F) Quantification of the ratio of p-DNAJB6b Y53 to DNAJB6b wild-type or DNAJB6b Y53F (E) and the ratio of p-SFKs Tyr416 to SRC, YES, FYN, or LYN (F) in (D) ( n =3). Data in (B-C, E-F) were expressed as mean ± SD, and p -values were calculated via unpaired two-tailed Student’s t -test. The individual replicate data in ( B ), ( C ), ( E ), and ( F ) are listed in Table S10 . (ns, not significant; ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: Primary antibodies (Document S1: Table S6 ) were used to detect tau (1:1000, GeneTex, Irvine, CA, USA), GFP (1:2000, GeneTex), Phospho-DNAJB6b (Tyr53) (1:250, this study), DNAJB6 (1:2000, Abcam, Cambridge, UK), V5 (1:1000, Abcam), AT8 (1:500, Thermo Fisher Scientific), Phospho-AKT (Ser473) (1:1000, Cell Signaling Technology), AKT (1:1000, Cell Signaling Technology), Phospho-Src Family (Tyr416) (1:500, Cell Signaling Technology), SRC (1:1000, Cell Signaling Technology), FYN (1:1000, GeneTex), Phospho-LYN (1:1000, Abcam), LYN (1:1000, GeneTex), YES (1:500, Cell Signaling Technology), HSPA8 (1:1000, Novus Biologicals, Centennial, CO, USA), HSPA1A (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), and β-actin (1:2000, Proteintech, Rosemont, IL, USA).

Techniques: Phospho-proteomics, Western Blot, Plasmid Preparation, Construct, Solvent, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Dephosphorylation of YES kinase-mediated co-chaperone DNAJB6b phosphorylation attenuates tau protein aggregation

doi: 10.1101/2025.11.14.688390

Figure Lengend Snippet: (A-L) Validation of SFKs responsible for the phosphorylation of DNAJB6b Y53. Knockdown of individual SFKs in SH-SY5Y cells was performed, including SRC ( A-C , n =3), FYN ( D-F , n =3), LYN ( G-I , n =3), and YES ( J-L , n =3), and analyzed by western blotting. (A, D, G, J) Immunoblotting with the indicated antibodies was used to assess the phosphorylation level of DNAJB6b and the knockdown efficiency of SFKs. β-actin served as a loading control. (B, E, H, K) Quantification of the ratio of p-DNAJB6b Y53 to endogenous DNAJB6b in shSRC (B) , shFYN (E) , shLYN (H) , or shYES (K) groups in (A, D, G, J) , respectively. (C, F, I, L) Quantification of the ratio of SRC (C) , FYN (F) , LYN (I) , or YES (L) to β-actin in (A, D, G, J) . (M) Assessment of YES-mediated phosphorylation at Y53 on His6-DNAJB6b wild-type by in vitro YES kinase assay. 2 nM recombinant His6-DNAJB6b wild-type proteins were incubated with YES kinase and ATP in the suggested tyrosine kinase reaction buffer at 30°C for 15 minutes. Immunoblotting with anti-p-DNAJB6b Y53 antibody and anti-p-SFK Tyr416 antibody assessed the phosphorylation level of His6-DNAJB6b and the activity of YES kinase, respectively. Coomassie blue staining confirmed that recombinant His6-DNAJB6b and YES kinase were equally loaded. (N) Quantification of the ratio of p-DNAJB6b Y53 to Coomassie blue-stained His6-DNAJB6b in ( M) ( n=3 ). Data in (B-C, E-F, H-I, K-L, N) were expressed as mean ± SD, and p -values were calculated via one-way ANOVA followed by Dunnett’s test. The individual replicate data in ( B ), ( C ), ( E ), ( F ), ( H ), ( I ), ( K ), ( L ), and ( N ) are listed in Table S10 . (ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: Primary antibodies (Document S1: Table S6 ) were used to detect tau (1:1000, GeneTex, Irvine, CA, USA), GFP (1:2000, GeneTex), Phospho-DNAJB6b (Tyr53) (1:250, this study), DNAJB6 (1:2000, Abcam, Cambridge, UK), V5 (1:1000, Abcam), AT8 (1:500, Thermo Fisher Scientific), Phospho-AKT (Ser473) (1:1000, Cell Signaling Technology), AKT (1:1000, Cell Signaling Technology), Phospho-Src Family (Tyr416) (1:500, Cell Signaling Technology), SRC (1:1000, Cell Signaling Technology), FYN (1:1000, GeneTex), Phospho-LYN (1:1000, Abcam), LYN (1:1000, GeneTex), YES (1:500, Cell Signaling Technology), HSPA8 (1:1000, Novus Biologicals, Centennial, CO, USA), HSPA1A (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), and β-actin (1:2000, Proteintech, Rosemont, IL, USA).

Techniques: Biomarker Discovery, Phospho-proteomics, Knockdown, Western Blot, Control, In Vitro, Kinase Assay, Recombinant, Incubation, Activity Assay, Staining