Anti hsp90 α β antibody

Cells were lysed in NP-40 lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% NP-40, and freshly supplemented with Halt Protease Inhibitor). Protein lysates were quantified by Bradford assay (Carl Roth). 10-30 µg of lysate were separated by SDS-PAGE, transferred onto Immobilon-P PVDF membrane (Merck-Millipore), and probed with an anti-HA antibody (3F10, Sigma-Aldrich). For loading controls, membranes were probed with anti-HSP90 α/β antibody (Santa-Cruz). Membranes were probed with anti-rat or anti-mouse horseradish peroxidase (HRP)-labelled secondary antibodies (Thermo Fischer Scientific; Cell Signaling respectively) and signals were visualized using an INTAS Advanced Fluorescence Imager (INTAS).

The antibodies and reagents used in this study were obtained from the following sources. Anti-IRS-1 antibody and anti-IRS-2 antibody for immunoprecipitation were raised in rabbits as described previously [47 (link)]. Anti-IRS-2 antibody for immunoblotting, anti-IGF-IR β antibody, anti-HSP90 α/β antibody and anti-ubiquitin antibody (P4D1) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-USP9X antibody was kindly provided by Dr. Stephen A. Wood (Griffith University, Queensland, Australia). Anti-USP9X antibody was also obtained from Abcam (Cambridge, MA, USA). Anti-FLAG antibody and anti-phosphotyrosine antibody (4G10) were from Sigma (St. Louis, MO, USA). Anti-Myc antibody (9E10) was from Millipore (Billerica, MA, USA). Anti-Akt antibody, anti-pAkt (Ser473) antibody, anti-Erk1/2 antibody, anti-pErk1/2 antibody and anti-IGF-IR β antibody were from Cell Signaling Technology (Dancers, MA, USA). MG132 was from Cell Signaling Technology (Dancers, MA, USA). Cycloheximide was from Nacalai Tesque (Tokyo, Japan). PD98059 was from Sigma (St. Louis, MO, USA), BMS754807 was from Funakoshi (Tokyo, Japan). WP1130 was from Life Sensors (Malvern, PA, USA). Recombinant human IGF-I was kindly gifted by Dr Toshiaki Ohkuma (Fujisawa Pharmaceutical Co., Osaka, current Astellas Pharma Inc., Tokyo, Japan).

Cells were lysed in NP-40 lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% NP-40, Halt Protease Inhibitor). Lysates were quantified by Bradford assay (Carl Roth). In general, 30 μg per sample were separated by SDS-PAGE, transferred onto PVDF membranes and probed with different primary antibodies. Endogenous SAMHD1 was probed with anti-SAMHD1 (3F5) antibody (novusbio); phosphorylated T592 was detected with a pT592-SAMHD1-specific antibody (ProSci). Myc-tagged proteins were probed with an anti-myc (9B11) antibody (Cell Signaling), FLAG-tagged proteins with anti-FLAG M2 antibody (Sigma), T7-tagged proteins with anti-T7 antibodies (Novagen, Abcam), and HA-tagged proteins with an anti-HA (16B12) antibody (Biolegend). To control for equal loading of cell lysates membranes were probed with anti-HSP90 α/β antibody (Santa Cruz). Subsequently, PVDF membranes were incubated with anti-mouse or anti-rabbit HRP-labeled secondary antibodies (Cell Signaling) and visualized using HRP substrate on an Intas Advanced Fluorescence Imager (Intas).