Anti caveolin 1

3-(4,5-dimethylthiazol-diphenyltetrazolium bromide (MTT), was purchased from Sigma Aldrich (Milan, Italy). The following rabbit polyclonal antibodies (Abs) were used: Anti-histone H3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-COX IV (1:250; Cell Signaling Technology) and anti-caveolin-1 (1:400; Cell Signaling Technology). The following mouse monoclonal Abs were used: anti-TRPML1 (clone F-10, 1:300 for Western blot, 1:50 for FACS analysis; Santa Cruz Biotechnology, Dallas, TX, USA), anti-TRPML2 (1:300 for Western blot; Santa Cruz Biotechnology), anti-LAMP1 (1:300; Santa Cruz Biotechnology), anti-calreticulin (1:1000; BD Biosciences, Milan, Italy) and anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH, 14C10, 1:1000; Cell Signaling Technology). anti-TRPML2 from Sigma Aldrich was used diluted 1:50 for FACS analysis.
The following secondary antibodies were used: horseradish peroxidase (HRP)-conjugated anti-mouse IgG and HRP-conjugated anti-rabbit IgG (1:2000; Cell Signaling Technology), FITC-conjugated anti-rabbit Ab (BD Biosciences), PE-conjugated anti-mouse Ab (BD Biosciences, Milan, Italy), Alexa Fluor-594-conjugated anti-mouse Ab (1:100; Invitrogen, San Diego, CA, USA), Alexa Fluor-488-conjugated anti-mouse Ab (1:100; Invitrogen) and Alexa Fluor-488-conjugated anti-rabbit Ab (1:100; Invitrogen).

Cells were washed once with ice cold PBS on ice, then lysed in 2x Laemmli Sample buffer, scraped and sonicated. All of the samples were boiled for 6 minutes at 100°C and analyzed by Western blotting. Protein samples were separated by 10% SDS-PAGE and transferred to 0.20 µM pore size nitrocellulose membrane with Trans-Blot^® Turbo™ Transfer System, 1.3 A, 25V for 10 minutes. The membranes were then blocked with 5% (w/v) nonfat dry-milk powder solution in TBS-Tween 20 (TBST) and incubated with specific primary antibodies overnight at 4°C. The primary antibodies used in this study were as follows: anti-Hsp90 (Cell Signaling Technology, Cat# 4874), anti-caveolin-1 (Cell Signaling Technology Cat# 3238), β-tubulin (BD Biosciences, Cat# 556321), anti-dynamin II (BD Biosciences, Cat# 610263). After incubation with the primary antibodies, the membranes were washed three times for 10 minutes with TBST and incubated with horseradish-peroxidase (HRP) conjugated anti-mouse (Cell Signaling Technology, Cat# 7076) or anti-rabbit (Cell Signaling Technology, Cat# 7074) secondary antibodies. Immunoreactive proteins were visualized by enhanced chemiluminescence (ECL) using autoradiography films. ImageJ software (Research Services Branch, National Institute of Health (Bethesda, MD) was used for densitometric analyses.