Molecular mechanisms of ARID5B
Description
Raw Western Blot images from three different experiments as listed below: 1- A V5 co-IP was performed in HAP1 cells overexpressing V5-GFP, full length, and domain mutants (delta ARID and delta BAH domains) of V5-ARID5B. Input as well as IPs were blotted for HDAC1 (Cell Signaling, 5356), HDAC2 (Cell Signaling, 57156), H3 (Abcam, Ab1791), and V5 (Thermo, R960-25). 2- NALM6 WT and ARID5B KO cells were stimulated with 10µ/mg Igµ for 10min. Expression of Erk (Cell Signaling, 4696), pErk (Cell Signaling, 4370), Akt (Cell Signaling, 2920) and pAkt (Cell Signaling, 4060) was probed for. 3- Protein levels of HDAC1 (Cell Signaling, 5356), HDAC2 (Cell Signaling, 57156), H3K27ac (Active Motif, 39133), H3K4me1 (Diagenode, C15410194) and H3 (Abcam, Ab1791) were assessed in NALM6 WT and ARID5B KO cells.
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Proteins were denatured using XT Sample Buffer (BioRad, #1610791) or 4x Laemmli Sample Buffer (BioRad, #1610747) for 10 min at 95°C. 4-12% Criterion XT Bis-Tris Protein Gels (BioRad, #3450125) or 4-20% Mini-Protean TGX Precast Protein Gels (BioRad, #4561096) were used to separate the proteins, which were then transferred to low-fluorescence PVDF membranes (BioRad, #1704275) using the High MW program of the Trans-Blot Turbo Transfer System (BioRad, #1704150). The membranes were blocked for 1 h at RT, prior to overnight primary antibody incubation, followed by 2 h RT secondary antibody incubation in EveryBlot blocking buffer (BioRad, #12010020). The BioRad ChemiDoc imager was used to develop the membranes. Antibodies used are listed below: HDAC1 (Cell Signaling, 5356) HDAC2 (Cell Signaling, 57156) H3 (Abcam, Ab1791) V5 (Thermo, R960-25) Erk (Cell Signaling, 4696) pErk (Cell Signaling, 4370) Akt (Cell Signaling, 2920) pAkt (Cell Signaling, 4060) Anti-Rabbit (Cell Signaling, 7074) Anti-Mouse (Cell Signaling, 7076) H3K27ac (Active Motif, 39133) H3K4me1 (Diagenode, C15410194) Protein ladder used: PageRuler Plus Prestained Protein Ladder (Thermo Fisher, 26619)