Ab76867

The unroofed cells were incubated in 3% bovine serum albumin/PBS (BSA, m/v, fresh, Fisher Bioreagents #BP9703) for 1.5 h, followed by primary antibody (1:100 in 3%BSA/PBS) incubation for 1 h. Next, cells were washed thoroughly with PBS and the secondary antibody tagged with fluorescence dye (1:500) or GFP-nanobody (1:500) was applied for 1 h. Afterwards, cells were washed 4 times in PBS and stored in fresh PBS at 4 C until the samples were imaged. The following antibodies were used: anti-Caveolin1-Rabbit (abcam #ab2910), anti-Caveolin1-mouse (Santa Cruz #sc-53564), anti-Cavin1-Rabbit (abcam #76919), anti-Cavin2-Rabbit (abcam #ab76867), anti-Cavin3-Rabbit (abcam #abcam2912), anti-EHD2-goat (abcam #ab23935), anti-Pacsin2-Rabbit (Proteintech #10518-2-AP), anti-EHBP1-Rabbit (Proteintech #17637-1-AP), anti-mouse-Clathrin heavy chain (Thermo-Fisher #MA1-065, 1:2000), anti-mouse-Dynamin2 (Santa Cruz, C-18; #sc-6400), anti-rabbit-Atto647N (Rockland #611-156-122), anti-goat-Atto647N (Rockland, #610-156-121), anti-goat-Atto647N (Rockland #605-456-013 S), anti-rabbit-Alexa568 (Invitrogen #A11036), Fab2-anti-rabbit-Alexa594 (ThermoFisher #A-11072), Fab2-anti-mouse-Alexa488 (ThermoFisher #A-11017), Fab2-anti-mouse-Alexa568 (ThermoFisher #A-11019), GFP-nanobody-Atto647N (Chromotek #gba647n-100), Phalloidin-Alexa488 (ThermoFisher #A12379).

The tumor samples and the paired normal samples in our hospital were patients suffering from LUAD or LUSC. Tissue sections were deparaffinized, rehydrated, and permeated using Triton X 100 (T8200, Solarbio, Beijing, China) and followed by antigen retrieval using EDTA Antigen Retrieval solution (c1034, Solarbio, Beijing, China). The sections were incubated with Anti-CAV1 antibody (ab32577, Abcam, UK), Anti-CAV2 antibody (ab79397, Abcam, UK), Anti-CAVIN1 antibody (ab48824, Abcam, UK), Anti-CAVIN2 antibody (ab76867, Abcam, UK), Anti-CAVIN3 antibody (ab179923, Abcam, UK) at 4 °C overnight followed by a biotinylated secondary antibody (diluted at 1:200) at RT for 60 min. Then, the sections were stained with DAB staining solution (AR1022, BOSTER Biological Technology, Wuhan, China). To quantitatively evaluate the expression levels of each protein in the samples from patients with LUAD and LUSC, we calculated the percentage of cells stained. The extent of staining was scored as the following criteria: (a) percentage of stained cells: 0 (0%), 1 (1%-25%), 2 (26%-75%), 3 (51%-75%), and 4 (> 75%); and (b) staining intensity: 0 (negative staining), 1 (low staining), 2 (medium staining), and 3 (high staining). The final scores were calculated by multiplying the scores of intensities with that of extent.

Cells were collected and washed with cold PBS and lysed on ice for 30 minutes using SDS lysis buffer supplemented with protease inhibitor cocktail (Roche, Switzerland). The collected protein was denatured in a 95°C water bath for 10 minutes. Equal amounts of proteins (30 ug) were separated using SDS‐PAGE. Then, proteins were transferred to PVDF membranes and blocked with 5% bovine serum albumin, followed by incubation with primary and secondary antibodies. The details of the antibodies used in this study are: Anti-SDPR (ab76867, abcam, USA), Anti-Cyclin A (sc-27168, Santa Cruz, USA), Anti-Cyclin B (sc-166210, Santa Cruz, USA), Anti-Cyclin D (sc-8396, Santa Cruz, USA), Anti-Cyclin E (sc-377100, Santa Cruz, USA), Anti-GAPDH (sc-47724, Santa Cruz, USA).