Rabbit anti zo 1

Western & IP cell lysis buffer (Beyotime, Shanghai, China) with PMSF (Amresco, Solon, Ohio, USA) was used to lyse tissues or cells for 30 min on ice following centrifugation at 12000 g for 10 min at 4 °C. BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) was employed to measure total proteins in the supernatants collected from the centrifugation. The equal amount of proteins were separated on 10% SDS-PAGE and transferred to a 0.45 μm PVDF membrane (Amersham Hybond, GE Healthcare, München, Germany) followed by blocking in 0.5% albumin from bovine serum (Amresco, Solon, Ohio, USA) overnight at 4 °C with specific antibodies. The primary antibodies used in the study were as follows: rabbit anti-ERp29, rabbit anti-pan-AKT and mouse anti-β-actin diluted at 1:2000 (Cell Signaling Technology, Danvers, MA, USA); rabbit anti-E-cadherin, rabbit anti-ZO-1, rabbit anti-snail, rabbit anti-Ser473-AKT, rabbit anti-Thr308-AKT, rabbit anti-GSK-3β, rabbit anti-phospho-GSK-3β(Ser9), rabbit anti-mTOR and rabbit anti-phospho-mTOR all diluted at 1:1000 (Cell Signaling Technology); rabbit anti-Vimentin diluted at 1:1500 (Abcam, ab92547). After 3 times washing in TBST for 10 min each time, the membrane was then incubated with the respective secondary antibodies at room temperature for 1 h and the immunoblot was developed through enhanced chemiluminescence (Lulong biotech, Xiamen China).

Cells or lung samples were collected and lysed in RIPA lysis buffer (50 mM Tris-HCl PH 7.4, 150 mM NaCl, 1% NP-40) containing both protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Fair Lawn, NJ, United States). Western blots were carried out as previously described using primary rabbit anti-ENT1 (Abgent, San Diego, CA, United States), rabbit anti-ENT2 (Abcam), rabbit anti-occludin, rabbit anti-phosphorylated FAK, rabbit anti FAK, rabbit anti ZO-1, rabbit anti claudin-1 (Cell signaling, Boston, MA, United States), mouse anti-CD73 antibodies (ThermoFisher Scientific), mouse anti-Hif-1α antibodies (Novus Biological, Littleton, CO, United States), or mouse anti-β-Actin antibodies (Sigma) and corresponding secondary antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA, United States). Finally, the membranes were developed with Pierce ECL Western Blotting Substrate (Thermo Fisher scientific, Fair Lawn, NJ, United States).

Immunofluorescence in tissues was performed as described previously^{16 (link),37 (link)–39 (link)}. Briefly, the skin tissues were embedded, fixed, permeabilized, incubated with primary mouse anti-K5 (1:200; Progen Biotechnik, Heidelberg, Germany), rabbit anti-Filaggrin (1:1000; Abcam, Cambridge, MA), rabbit anti-E-cadherin (1:500; Cell Signaling, Danvers, MA), mouse anti-β-catenin (Cell Signaling, Danvers, MA), rabbit anti-F-actin (Cell Signaling, Danvers, MA) and rabbit anti-ZO-1(Cell Signaling, Danvers, MA), and then incubated with Alexa Fluor 488 F (ab’) 2 fragments of goat anti-guinea pig IgG antibodies and Alexa Fluor 568 of goat anti-rabbit IgG antibodies (Invitrogen, Carlsbad, CA). The cells were then fixed in Prolong Gold Antifade with DAPI (Invitrogen, Carlsbad, CA) to visualize the cell nuclei, and observed under a fluorescence microscope (OlympusIX71, Japan) with a peak excitation wavelength of 340 nm.