Anti cbl

PMA (phorbol 12-myristate 13-acetate), ATP, nigericin, CCCP, PP2, dAdT, and MG132 were purchased from Sigma. MitoSox, H₂-DCFDA, TMRE, MitoTracker Green FM, and Hoechst were purchased from Life Technologies. MitoTEMPO was purchased from Enzo Life Sciences. Hydrocotarnine was purchased from Enamine. Anti-Pyk2, anti-AIM2, and anti-p-Pyk2 were purchased from Cell Signaling. Anti-Cbl, anti-ASC, anti-caspase-1, anti-IL-1β, anti-phosphotyrosine, and anti-GAPDH were purchased from Santa Cruz. Anti-Ly6G, anti-CD45, and anti-CD11b were purchased from BD Bioscience. Anti-NLRP3 and anti-F4/80 were purchased from BioLegend and eBioscience, respectively. Plasmids encoding mutants CBL (Y371D) and CBL (Y371F) were generated by ligating amplified DNA fragments into the NheI/PmeI-treated pLKO_AS2.neo vector (RNAi Core, Taiwan). The mutant CBL was constructed using a QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies), and the following primers (forward and reverse, respectively): for Cbl Y371D, 5′-CAGGAACAATATGAATTAGACTGTGAGATGGGCTCCAC-3′ and 5′-GTGGAGCCCATCTCACAGTCTAATTCATATTGTTCCTG-3′; and for Cbl Y371F, 5′-CAGGAACAATATGAATTATTCTGTGAGATGGGCTCCAC-3′ and 5′-GTGGAGCCCATCTCACAGAATAATTCATATTGTTCCTG-3′.

Anti-EGFR, anti-Cbl and anti-endophilins antibodies were purchased from Santa Cruz. Anti-CIN85 monoclonal antibody was from Upstate. Anti-flag monoclonal antibody, phosphatidic acid, DMP (dimethylpimelimidate) and horse radish peroxidase-conjuated secondary antibodies were from Sigma. EGF, Alexa Fluor 647 labeled EGF, PIP Strips™ membrane and fluorescence labeled secondary antibodies (Alexa Fluor 488/546 conjugates) for immunofluorescence were the products of Life Technologies. The leica laser scanning confocal microsystem, including the leica TCS SP2 confocal microscope, Leica confocal scanner, and Leica confocal acquisition software were used with the HCX PL APO 1bd. BL 63.0 X/1.4 oil objective at 1.4 numerical aperture at a working temperature of 22°C.

Sigma Chemical Co. provided honokiol, cycloheximide and MG132 (St. Louis, MO, USA), and R&D system supplied recombinant human recombinant TRAIL and z-VAD-fmk (Minneapolis, MN, USA). Enzo Life Sciences provided lactacystin (Ann Arbor, MI, USA). The primary antibodies were as follows: Cell Signaling Technology supplied anti-PARP, anti-cleaved caspase-3, anti-Bcl-xL, anti-DR5, anti-CHOP, and anti-UCHL1 (Beverly, MA, USA). Sigma Chemical Co. supplied anti-actin (St. Louis, MO, USA). Enzo Life Sciences provided anti-pro-caspase-3 and anti-c-FLIP (San Diego, CA, USA). BD Biosciences provided anti-Bim and anti-XIAP (San Jose, CA, USA). Abcam supplied anti-DR4 (Cambridge, MA, USA). R&D system supplied anti-survivin (Minneapolis, MN, USA). Santa Cruz Biotechnology provided anti-Mcl-1, anti-Bcl-2, anti-cIAP2, anti-ATF4, anti-Ub, anti-Cbl, anti-Itch, anti-USP14, anti-USP33, anti-OTUB1, anti-TRABID, and anti-STAMBPL1 (St. Louis, MO, USA). Bethyl Laboratories Inc provided anti-USP7 and anti-USP8 (Montgomery, TX, USA). Novus Biologicals supplied anti-USP53 (Centennial, CO, USA). Abnova provided anti-USP9X (Taipei City, Taiwan). The siRNAs were as follows: GFP (control) siRNA (Bioneer, Daejeon, Korea), DR5 siRNA (Invitrogen, Carlsbad, CA, USA), and STAMBPL1 siRNA (Santa Cruz Biotechnology, St. Louis, MO, USA). STAMBPL1 plasmid was a gift from Dr. H.C. Kang (Ajou University, Suwon, Korea).

FcγR1 specific antibody [mAb 32.2 F(ab)₂ frgment for IgG] was purchased from Medarex (Annandale, NJ). The cross linking Ab was a rabbit anti-mouse F(ab)₂ fragment (R/M) obtained from Organon technika (West Chester, PA). Sheep red blood cells and rabbit IgG fraction to sheep RBC were purchased from ICN (Costa Mesa, CA). Anti-phosphotyrosine (4G10) was procured from Upstate Biotechnology (Lake Placid, NY). Anti-CBL and anti-CRKL were obtained from Santa Cruz biotechnology (Santa Cruz, LA). PKC activator, PMA (phorbol ester) and PKC inhibitor bisindolymaleiamide 1 (GF109203X) were from Calbiochem (San Diego, CA).