Rabbit anti cav 1

Mouse anti-CD44 (clone 5035-41.1D, Novus Biologicals), rabbit anti-CAV1 (BD Biosciences), rabbit anti-Caveolin2 (Sigma Aldrich), rabbit anti-Cavin-1 and Cavin-4 antibody were raised as described previously [16] (link), rabbit anti-Cavin-3 (ProteinTech group), mouse anti-Cavin-2 (Sigma Aldrich), rabbit anti-HA (Sigma Aldrich), mouse anti-Cdc42 (Becton Dickinson), mouse anti-GFP (Roche), mouse anti-transferrin receptor antibody (Zymed), Alexa Fluor conjugated dextran (Life Technologies), anti-rabbit and anti-mouse Alexa Fluor antibodies (Invitrogen), Alexa Fluor conjugated transferrin (Invitrogen), Alexa Fluor conjugated phalloidin (Invitrogen), Filipin III (Sigma Aldrich), Dynasore (Sigma Aldrich), 7-Ketocholesterol (Sigma Aldrich), Protease Inhibitor Cocktail Set III (Merck Millipore), PhosSTOP Phosphatase Inhibitor Cocktail (Roche), Dyngo-4a (Sigma). Stealth RNAi siRNA duplex oligonucleotides targeted against mouse Cavin-1 (5′CCGCUGUCUACAAGGUGCCGCCUUU3′;5′AAAGGCGGCACCUUGUAGACAGCGG3′) and Cavin-3 (5′CCGGAGCUCUGAAGGCCCAUCAGAA3′; 5′UUCUGAUGGGCCUUCAGAGCUCCGG3′) (Invitrogen).

Cells lysates were prepared in RIPA buffer plus 1:100 protease inhibitor cocktail, phosphatase inhibitor cocktail 2, and phosphatase inhibitor cocktail 3 (all Sigma-Aldrich), and the protein concentration was determined by BCA assay (Thermo Scientific™ Pierce™ Protein Biology, Rockford, IL, USA). Equal volumes of protein (1 or 5 μg) were electrophoresed by SDS-PAGE in a 12 % polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) blots which were blocked in 5 % milk or 5 % bovine serum albumin (BSA; Sigma-Aldrich) in Tris-buffered saline + 0.05 % Tween 20 (TBST), and then incubated overnight at 4 °C with either rabbit anti-Akt or anti-serine 473 phosphorylated Akt (p-Akt) (1:1000 in TBST + 5 % BSA; both Cell Signaling Technology), or rabbit anti-Cav-1 (1:5000 in TBST + 2.5 % milk; BD Biosciences, Franklin Lakes, NJ, USA), or rabbit anti-GM130 (1:1000 in TBST + 5 % BSA; Cell Signaling Technology). Secondary antibodies were Amersham ECL donkey anti-rabbit or sheep anti-mouse horseradish peroxidase (HRP)-linked IgG antibody (both GE Healthcare UK Limited, Little Chalfont, UK). HRP activity was detected using Supersignal West Dura, Extended Duration Substrate (Thermo Scientific™ Pierce™ Protein Biology) and the chemiluminescence reaction was visualized using a FOTO/Analyst® Fx CCD imaging system (Fotodyne Inc., Hartland, WI, USA).