Anti actin a2066

Lysates of the patients P1 and P2 and control fibroblasts as well as HeLaM cells were used for immunoblot analysis with anti-human VPS11 (WH0055823M1; Sigma-Aldrich), anti-human VPS41 (sc-377 118; Santa Cruz) or antibodies to VPS33A, VPS 18, HA and actin as described previously (10 ). In addition, lysates from the transfected HeLaM cells were subjected to immunoblot analysis with anti-HA (HA.11, MMS-101R; Covance) and anti-actin (A2066; Sigma-Aldrich) antibodies. In experiments to examine the effects of the proteasome inhibitor MG-132 on HeLaM cells stably expressing VPS33A^WT-HA or VPS33A^R498W-HA, immunoblotted VPS33A bands were quantified by densitometry using ImageJ software, normalized to actin bands and fold changes in VPS33A concentration calculated relative to no incubation with MG-132. In each separate experiment, the effect of MG-132 on both VPS33A^WT-HA and VPS33A^R498W-HA was measured. A paired student t-test was used to calculate P-values.

Retro-2 [2,3-Dihydro-2-(5-methyl-2-thienyl)-3-phenyl-4(1H)-quinazolinone], vinblastine and rabbit anti-actin (A2066) were purchased from Sigma-Aldrich. LysoTracker Red^®, DQ^TM Red BSA, and 4′,6-diamidino-2-phenylindole (DAPI) were from Invitrogen Life Technologies. Anti-α-tubulin mAb (11H10) was from Cell Signaling Technology (Ozyme, Saint Quentin en Yvelines, 78053, France). Anti-actin (A2066) was from Sigma-Aldrich (Saint Quentin Fallavier, 38070, France). Anti-human cathepsin D (E-7) was from Santa Cruz Biotechnology Inc. (CliniSciences, Nanterre 92000, France). Anti-LC3 antibody (L7543), anti-β-actin-peroxydase (A3853), goat anti-rabbit IgG- and sheep anti-mouse IgG-peroxidase antibodies (A0545 and A5906, respectively) were from Sigma-Aldrich. Appropriate secondary antibodies for indirect immunofluorescence labeling were purchased from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA, United States) and Molecular Probes Inc. (Invitrogen Life Technologies).

Cells were plated onto 10 cm dishes at a density of 1 × 10⁵ cells/mL in the presence of various concentrations of reagents. After incubation for indicated durations, cells were collected and washed twice with PBS (−). Cell protein was extracted and Western blot analysis was done as described previously [23 (link)]. A c-Abl (sc-23) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-actin (A2066) was obtained from Sigma-Aldrich. Then, p44/42 MAPK (Erk1/2), phospho-p44/42 MAPK (Thr202/Tyr204), AKT, phospho-AKT (Ser473), and caspase-3 antibodies were from Cell Signaling Technology Japan (Tokyo, Japan). Anti-PARP antibody was from WAKO Chemicals (Osaka, Japan).

Antigen affinity-purified sheep anti-SUMO-1 antibody was a kind gift from Professor Ron Hay (Dundee) (IB: 1:2,000). Anti-Parkin mouse monoclonal was obtained from Santa Cruz (sc-32282) (IB: 1:5,000; IF: 1:1,000); anti-FLAG HRP (A8592) (IB: 1:10,000) and anti-actin (A2066) (IB: 1:5,000) antibodies were obtained from Sigma; anti-CISD1 (16006-1-AP) (IB: 1:1,000) and anti-TOMM70A (14528-1-AP) (IB: 1:1,000) antibodies were obtained from Proteintech Europe; and anti-ubiquitin antibody (Z0458) (IB: 1:1,000) was purchased from Dako. Epitomics raised anti-Parkin phospho-serine 65 rabbit monoclonal antibody in collaboration with the Michael J Fox Foundation for Research (IB: 1:1,000; IF: 1:500).

For Western blot analysis, protein samples were separated by SDS-PAGE, transferred to a nitrocellulose membrane (0.2 mm pore size; Bio-Rad, Hercules, CA, USA). The blots were probed with the primary antibodies listed below and bands were detected by horseradish peroxidase (HRP)-conjugated secondary antibody (Bio-Rad). Peroxidase activity was detected with the enhanced chemiluminescence detection method (WesternBright ECL, Advasta, Menlo Park, CA, USA). Primary antibodies used in this study were: rabbit polyclonal anti-ubiquitin antibody (kindly provided by Prof. A. L. Haas, Dept. of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans); anti-HSF2 (sc-13056), anti-YY1 (sc-281), anti-specificity protein 1 (Sp1) (sc-420) and anti-specificity protein 3 (Sp3) (sc-644) from Santa Cruz Biotechnology (Dallas, TX, USA); anti-Lamin A/C (4C11) and anti-HSF1 (4356) from Cell Signaling Technology (Danvers, MA, USA); anti-GAPDH (A300-641A) from Bethyl Laboratories, Inc.; anti-actin (A 2066) from Sigma-Aldrich (Steinheim, Germany).