resource source identifier goat anti-cyct1 santa cruz sc-8127  (Santa Cruz Biotechnology)

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: P‐TEFb interacts with CRTC2 and enhances CRTC2‐dependent transcription. a) Immunofluorescence analysis of the co‐localization of CRTC2 and CycT1 in HeLa cells treated with DMSO or FSK (left). Line scans along the dotted lines in the images (right). b) Co‐IP assay examining the interaction between endogenous CRTC2 and P‐TEFb in 293T cells treated with DMSO or FSK for 1 h. c) Co‐IP assay examining the interactions between FLAG‐CDK9 and HA‐CRTC2 in 293T cells treated with the indicated concentration of FSK or H89. d) Co‐IP assay examining the interactions between FLAG‐CDK9 and HA‐CRTC2 in 293T cells ectopically expressing Myc‐SIK1. e) Co‐IP assay examining the interactions between FLAG‐CDK9 and HA‐CRTC2 (WT or S171/274A mutants) in 293T cells. f) Quantification of CRE‐luc luciferase activity in 293T cells transfected with the indicated amounts of FLAG‐CDK9 plasmids and treated with DMSO or FSK (10 µ m ) for 6 h. g) Quantification of CRE‐luc luciferase activity in 293T cells treated with flavopiridol (FP, 300 n m ) and FSK (10 µ m ) for 6 h. h) Quantification of CRE‐luc luciferase activity in HA‐CRTC2‐expressing 293T cells infected with lentivirus carrying sgRNA against CDK9 (sgCDK9) or GFP (sgControl) and treated with DMSO or FSK (10 µ m ) for 6 h. i) Quantification of CRE‐luc luciferase activity in FLAG‐CDK9‐expressing 293T cells infected with lentivirus carrying sgRNA against CRTC2 (sgCRTC2) or GFP (sgControl) and treated with DMSO or FSK (10 µ m ) for 6 h. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. **** p < 0.0001; n.s., not significant. All results are from more than three independent experiments.

Article Snippet: One day after transfection, cells were excited with a 488 nm laser for 5 min. Then incubated with primary antibody against CDK9 (Santa Cruz, sc‐13130) or CycT1 (Santa Cruz, sc‐8127).

Techniques: Immunofluorescence, Co-Immunoprecipitation Assay, Concentration Assay, Expressing, Luciferase, Activity Assay, Transfection, Infection

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: CRTC2 extracts P‐TEFb from HEXIM1‐containing inhibitory complex. a) Live‐cell imaging of 293T cells co‐expressing CDK9‐mCherry and CRTC2‐eGFP or R>A‐eGFP and treated with DMSO or FSK (10 µ m ) for 1 h (left). The dotted line area indicates the nucleus. Quantification of cells with CDK9‐mCherry foci upon treatment with DMSO or FSK (right). b) Immunofluorescence analysis of co‐condensates of CRTC2‐IDR‐mCherry‐CRY2 with CDK9 or CycT1 in 293T cells before or after blue light stimulation (left). Quantification of cells with CRTC2‐IDR co‐condensates with CDK9 or CycT1 (right). c) Live‐cell imaging of 293T cells co‐expressing CycT1‐CFP, HEXIM1‐RFP, CRTC2‐eGFP, and R>A‐eGFP as indicated. d) Quantification of cells containing CycT1‐CFP foci. e) RNA‐IP assay examining the association between FLAG‐CDK9 and 7SK snRNA in 293T cells co‐expressing HA‐CRTC2 or HA‐R>A and treated with DMSO or FSK. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. **** p < 0.0001. All results are from more than three independent experiments.

Article Snippet: One day after transfection, cells were excited with a 488 nm laser for 5 min. Then incubated with primary antibody against CDK9 (Santa Cruz, sc‐13130) or CycT1 (Santa Cruz, sc‐8127).

Techniques: Live Cell Imaging, Expressing, Immunofluorescence

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: CRTC2 activates cystogenesis‐associated genes by promoting paused Pol II release. a) Immunofluorescence analysis of CRTC2 and CycT1 co‐localization in the kidneys of WT and Pkd1 −/‐ mice ( n = 3). b) Immunofluorescence analysis of CRTC2 and CycT1 co‐localization in WT 9–12 cells (left). Line scan of the dashed line in the merged inset image (right). c) Western blot analysis of CRTC2 expression in WT 9–12 cells transfected with the indicated sgRNAs and constructs. d) Immunofluorescence analysis of ectopically expressed TY1‐CRTC2 and TY1‐CRTC2‐R>A in WT 9–12 cells with endogenous CRTC2 knocked out. e) RT‐qPCR analysis of representative CRTC2‐target genes in WT 9–12 cells transfected with the indicated sgRNAs and constructs. f) Cumulative curve of Pol II pausing index (PI) for CRTC2 target genes in normal and ADPKD kidney tissues. g) ChIP‐qPCR analysis of CycT1, Pol II Ser2P, and Pol II occupancy on LDHA and PLAU genes in WT 9–12 cells with endogenous CRTC2 replaced by TY1‐CRTC2 or TY1‐CRTC2‐R>A mutant. The inset bar graphs indicate the Pol II pausing index for LDHA and PLAU genes, respectively. h) Schematic illustration of how CRTC2 triggers the productive elongation of cystogenesis‐associated genes, ultimately leading to ADPKD progression. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar, 20 µm (a,b). All results are from more than three independent experiments.

Article Snippet: One day after transfection, cells were excited with a 488 nm laser for 5 min. Then incubated with primary antibody against CDK9 (Santa Cruz, sc‐13130) or CycT1 (Santa Cruz, sc‐8127).

Techniques: Immunofluorescence, Western Blot, Expressing, Transfection, Construct, Quantitative RT-PCR, ChIP-qPCR, Mutagenesis