Alexa fluor conjugated transferrin

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).

Indirect immunofluorescence was performed after paraformaldehyde fixation and permeabilization with 0.1% Triton X-100 or 0.5% saponin. For imaging of transferrin endocytosis, the living cells were first serum-starved for 30 min, followed by exchange for medium supplemented with Alexa Fluor-conjugated transferrin (Invitrogen) for the indicated times. For TIRF microscopy, HEK293 cells were cultured and transfected using CALM-GFP and AP2 μ2-mCherry as detailed previously (Gage et al., 2005; Taylor et al., 2011 ). Cells were plated on clean coverslips on the same day as transfection and imaged 48 hr later to allow several cell divisions and to give transfected cells time to adapt moderate protein overexpression (Taylor et al., 2011, 2012 ). To determine CCP maturation time in CALM knockdown cells, HT1080 cells were transfected with either CALM or control siRNA followed by transfection with CALM-GFP and AP2 μ2-mCherry. The maturation time of CCPs was determined as described previously. For STED super resolution microscopy, samples were fixed and processed as described above. For microscopy, a Leica TCS SP8 gSTED equipped with a 100×/1.4 Oil STED Orange lens and a 592 nm depletion laser was used. Further details on STED imaging and quantification are summarized in the Supplemental Experimental Procedures.