p6344 pcdna4 to ha brd4fl  (Addgene inc)

Journal: bioRxiv

Article Title: The ESCRT protein CHMP5 promotes T cell leukemia by controlling BRD4-p300-dependent transcription

doi: 10.1101/2024.01.29.577409

Figure Lengend Snippet: (A) Western blot of fractionated human T cells isolated from PBMCs. W, whole cell; C, cytoplasmic; and N, nuclear lysates. CHMP5 band intensity relative to the cytoplasmic band are indicated. (B) Clustal Omega alignment of the N-terminal amino acid sequences of CHMP5 from different species. Sequences corresponding to the bipartite nuclear localization sequence (NLS) are highlighted. (C) Amino acid sequence homology of CHMP5 from different species relative to human CHMP5. (D) Western blot of fractionated CUTLL1 cells transduced with CHMP5-HA and subjected to immunoprecipitation with isotype (IgG) or anti-HA antibodies and immunoblotted for MYC and ICN1. (E) Western blot of nuclear lysates in LOUCY cells subjected to immunoprecipitation with IgG or anti-BRD4 antibodies (F) Nuclear lysate of HEK293T cells co-transfected with BRD4-FLAG or CHMP5-HA and subsequently immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-HA or anti-FLAG antibodies. (G) Anti-FLAG immunoprecipitation of recombinant BRD4-FLAG and CHMP5-His recombinant protein. (H) PCR of RPL30 on antibody (Ab) immunoprecipitated chromatin DNA from CUTLL1 cells transduced with CHMP5-HA run on a 1% agarose gel.

Article Snippet: BRD4 overexpression plasmid for HEK293T transfection was purchased from Addgene (p6344 pcDNA4-TO-HA-Brd4FL #31351). shRNA constructs in a pLKO.1 vector targeting human CHMP5 were purchased from Sigma Aldrich along with 2 control non-targeting shRNAs. shRNAs targeting human MYC were a gift from Xi Chen (Baylor College of Medicine). shRNA sequences can be found in .

Techniques: Western Blot, Isolation, Sequencing, Transduction, Immunoprecipitation, Transfection, Recombinant, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: The ESCRT protein CHMP5 promotes T cell leukemia by controlling BRD4-p300-dependent transcription

doi: 10.1101/2024.01.29.577409

Figure Lengend Snippet: (A) Relative CHMP5 protein expression in cancer cell lines in order from highest to lowest average. Data from Cancer Cell Line Encyclopedia (CCLE). (B) Western blot of T-ALL cell lines and healthy donor human T-cells. Lysates from HEK293T (HEK) cells transduced with non-targeting shRNA (CT) or shCHMP5 (KD) lentivirus is used as control for anti-CHMP5 antibody specificity. (C) Quantification of CHMP5 and VPS4 protein relative to Actin from (B). Student’s t-test: ****, p < 0.0001. (D-E) Overall survival of pediatric T-ALL patients (TARGET T-ALL) expressing high (top 20%) and low (bottom 20%) levels of VPS4A (D) and CHMP1A (E). (F) mRNA expression of CHMP5 in pediatric T-ALL patients that achieved complete remission (CR) (n = 65) or did not achieve complete remission (NCR) (n = 4). Student’s t-test: *, p < 0.05. (G) Expression of BIM in CUTLL1 cells from . Data are mean (± SD) of technical replicates. One-way ANOVA: ****, p < 0.0001 (H) Viability of CT and KD CUTLL1 and LOUCY treated with JQ1 for 3 days. Data are presented as mean (± SD) of 3 technical replicates, representative of 2 independent experiments. (I) IC 50 for JQ1 in CUTLL1 and LOUCY cells. IC 50 calculated by non-linear best-fit analysis. p-value calculated by 2-way ANOVA. FC, fold-change in CT versus KD IC 50 .

Article Snippet: BRD4 overexpression plasmid for HEK293T transfection was purchased from Addgene (p6344 pcDNA4-TO-HA-Brd4FL #31351). shRNA constructs in a pLKO.1 vector targeting human CHMP5 were purchased from Sigma Aldrich along with 2 control non-targeting shRNAs. shRNAs targeting human MYC were a gift from Xi Chen (Baylor College of Medicine). shRNA sequences can be found in .

Techniques: Expressing, Western Blot, Transduction, shRNA, Control