Anti phospho crkl

Spautin-1 (S7888), Imatinib (S1026), and MG132 (S2619) were from Sellectchem (Houston, TX, USA). SKP2-C25 (M60136) was from Xcessbio Biosciences, Inc. (San Diego, CA). MTS reagent (G3582) was from Promega Corporation (Madison, WI, USA). Co-IP assay kit (14311D) was from Life Technologies (Carlsbad, CA). Antibodies: anti-Ubiquitin (#3936), anti-K63-Ubiquitin (#12930), anti-K48-Ubiquitin (#12805), anti-USP10 (#8501), anti-USP1 (#8033), anti-USP2 (#8036), anti-USP7 (#4833), anti-USP8 (#11832), anti-USP14 (#11931), anti-USP15 (#66310), anti-USP18 (#4813), anti-UCHL1 (#13179), anti-UCHL3 (#8141), anti-CYLD (#8462), anti-A20 (#5630), anti-SKP2 (#2652), anti-p27 (#3686), anti-FLAG (#14793), anti-HA (#3724), anti-phospho-c-Abl(Y245) (#2861), anti-c-Abl (#2862), anti-phospho-STAT5 (#9359), anti-STAT5 (#25656), anti-phospho-Crkl (#3181) and anti-Crkl (#38710) (Cell Signaling Technology, Beverly, MA, USA); anti-UCHL5 (ab124931), anti-USP13 (ab109264) (Abcam, Cambridge, MA); anti-GAPDH (BS60630) (Bioworld Technology, Inc., Louis Park, MN, USA).

Cells were lysed in Laemmli buffer, lysates clarified by centrifugation (20,000 g, 10 min, room T) and protein concentration in supernatants determined using the BCA method. Equal protein aliquots (50 mg) were subjected to SDS-PAGE in 9% polyacrylamide gel and then electroblotted onto nitrocellulose membranes (GE Healthcare Life Sciences; Pittsburgh, PA, USA). Membranes were incubated for 1 h at RT in PBS/0.1% BSA (Sigma-Aldrich) and then overnight at 4 °C with the primary antibody in PBS/0.1% BSA/0.1% Tween-20. Primary antibodies (Ab) used were rabbit polyclonal anti-c-Abl (sc-131, Santa Cruz Biotechnology; St. Cruz, CA, USA), diluted 1:750 in PBS/0.1% Tween-20; rabbit polyclonal anti-phospho-Crkl (#3181, Cell Signaling Technology; Danvers, MA, USA), rabbit anti-ARD1 (produced in Dr. Nathalie Mazure’s laboratory) and mouse monoclonal anti-vinculin (V9131, Sigma-Aldrich), all diluted 1:1000 in PBS/0.1% Tween-20. After washing three times with PBS/0.1% Tween-20, membranes were incubated for 1 h at RT in PBS/0.1% BSA containing an IRDye800CW- or IRDye680-conjugated secondary Ab (LI-COR^®; Lincoln, NE, USA). Mouse and rabbit IRDye800CW-Ab were diluted 1:20,000, goat IRDye680-Ab 1:40,000, mouse and rabbit IRDye680-Ab 1:30,000, goat IRDye680-Ab 1:15000. Ab-coated protein bands were visualized using Odyssey Infrared Imaging System Densitometry (LI-COR^®).

Ponatinib was purchased from Shanghai Biochempartner Co., Ltd. (Shanghai, China). The HDAC inhibitor vorinostat (suberoylanilide hydroxamic acid) was provided by Merck & Co (New Jersey, NJ). Stock solutions of vorinostat and ponatinib were dissolved in dimethyl sulfoxide (DMSO) and subsequently diluted to the desired concentration in the growth medium. Anti-phospho Abl, anti-phospho Crk-L, anti-cleaved caspase 3, anti-poly (ADP-ribose) polymerase (PARP), and anti-acetyl-histone H4 antibodies were purchased from Cell Signaling (Beverly, MA). β-Tubulin and β-actin antibodies were provided by Santa Cruz Biotechnology (Dallas, TX). Other reagents were obtained from Sigma (St Louis, MI).