Anti desmoplakin

Anti–α–actinin-2 was from Abcam [ab9465 (1:500) and ab137346 (1:200)] and Fitzgerald (70R-1068; 1:200). Anti-PKP2 was from SLBio (LS-C85371; 1:200). Anti-desmoplakin was from Abcam (ab71690; 1:200). Anti-Cx43 was from Abcam (ab11370; 1:200). Anti–N-cadherin was from Cell Signaling Technology (#14215; 1:100).
CMs cultured in growth medium were perm/fixed with 0.03% Triton X-100 (Thermo Fisher Scientific) and 1% paraformaldehyde (Thermo Fisher Scientific) in phosphate-buffered saline (PBS) containing calcium and magnesium (PBS⁺) at 37°C for 90 s. Cells were immediately postfixed in 4% paraformaldehyde in PBS⁺ at 37°C for 15 min. Cells were rinsed three times with PBS⁺ and permeabilized with 0.5% Triton X-100 in PBS⁺ for 10 min. Cells were blocked with 2% bovine serum albumin (BSA; Sigma-Aldrich) in PBS⁺. Primary and secondary antibodies were applied in 2% BSA in PBS⁺ and rinsed three times over 30 min with PBS⁺ between each treatment. For Western blot analyses, CMs were lysed in 2× NuPAGE LDS Sample Buffer (Life Technologies) containing 100 mM dithiothreitol and analyzed by SDS–polyacrylamide gel electrophoresis and immunoblotting with chemiluminescent horseradish peroxidase detection. Immunofluorescent images and Western blots were adjusted for brightness and contrast using ImageJ software (National Institutes of Health, Bethesda, MD).

Anticathepsin D, anti–c-Jun, and anti–Egr-1 were from Santa Cruz; anti–Iba-1 was from Wako; anti-CD44 was from Cell Signaling; antivimentin was from Dako; anti-GAPDH, antidesmoplakin, anti– HMOX-1, anti-Prdx6, anti- ALDH1A1, and anti- IGF1 were from Abcam; antiheat-shock protein 27 (hsp27) was from Novus; anticlusterin was from Chemicon, anticystatin C was from Upstate; and anti–α-actin was from Sigma.