GTF2H4 Antibody - #DF8857

Figure 4. Analysis of the effect of HMGA1 on the combination of RNAPII and TFIIH in HGPS cells A. Interaction of endogenous HMGA1 with HMGA2 and TFIIH4 complex was analyzed by Co‐IP assay using anti‐HMGA1 antibody in H1299 cells. Pulled‐down protein complexes were analyzed. Benzonase (B) was added in cell lysate. B. Interaction of endogenous HMGA2 with HMGA1 and TFIIH4 complex was analyzed by Co‐IP assay using anti‐HMGA2 antibody in H1299 cells. Pulled‐down protein complexes were analyzed. C. HMGA1‐associated proteins from HEK293T cells expressing FLAG‐HMGA1 were IP with anti‐Flag antibody. The protein bands were analyzed by mass spectrometry. Representative peptide fragments of HMGA1 associated proteins and peptide coverage of the indicated proteins were shown. D. Interaction of endogenous CDK7 with HMGAs, XPB and TFIIH4 was analyzed by Co‐IP assay using anti‐CDK7 antibody in H1299 cells. Pulled‐down protein complexes were analyzed. E‐F. HEK293T cells were transfected with Flag‐HMGA1 (E) or CDK7 (F). HMGA1 or CDK7 was IP with anti‐Flag antibody and anti‐CDK7 antibody. G. HEK293T cell lysates were incubated with GST or GST‐HMGA1 sepharose beads. Pulled‐down protein complexes were analyzed. H. Scheme of HMGA1 wild‐type structure, including AT hooks (AT) and acidic tail (C tail), and deletion mutants used for immunoprecipitation experiments below. I. HEK293T cells were transfected with Flag‐HMGA1 deletion mutants. IP of Flag was performed. Pulled‐down protein complexes were analyzed. J. HEK293T cell lysates were incubated with GST or GST‐HMGA1 deletion mutants sepharose beads. Recombinant proteins from pull‐down assays visualized by silver staining and Western blot. K, L. Progerin‐expressing H1299 cells were transfected with His‐control or His‐HMGA1. Immunoprecipitation of RNA Pol II (K) or CDK7 (L) with anti‐RNAPII antibody or anti‐CDK7 antibody was performed, respectively. Pulled‐down protein complexes were analyzed. M, N. RNAPII, the RNAPII Ser2P, and RNAPII Ser5P in HGAFDFN003 cells (M) and 88, 92‐year‐old cells (N) compared with CRL‐1474 cells were detected by Western blot. O. Progerin‐expressing CRL‐1474 cells were transfected with His‐control or His‐HMGA1. Indicated proteins and modifications were detected by western blot. P. Infection with siCtrl or siCDK7 in Flag‐HMGA1‐transfected HGPS cells (HGAFDFN003), cyclin A1, RNAPII, the RNAPII Ser2P and RNAPII Ser5P was detected. Q. Infection with control shRNA (shCtrl) or HMGA1 shRNA (shHMGA1) in anti‐miR‐59‐transfected HGAFDFN003 cells. Indicated proteins and modifications were detected by western blot. Source data are available online for this figure.