Polyclonal goat anti rabbit antibody

Relative amounts of aquaporin 2 (AQP2) were validated by Western blot analyses, essentially as previously described [19 (link)]. The urine samples were separated on Criterion TGX Stain-free AnykD SDS-PAGE gels (Biorad). The primary antibody against Anti-Water Channel AQP2 was produced in rabbits (A7310 from Sigma-Aldrich), and the secondary antibody was polyclonal goat anti-rabbit antibody (DAKO, Glostrup, Denmark). Chemiluminescence and fluorescence detection of the membranes were performed to quantitate AQP2 and total protein stain (from the stain-free gels), respectively. ECL-kit (Thermo Scientific) was used according to the manufacturer’s recommendations, and the membranes were scanned on ImageQuant LAS 4000 (GE Healthcare). Quantification was performed in ImageQuant TL 7.0 (GE Healthcare).

Preparation of extract of extracellular matrix of cultured keratinocytes, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transfer to nitrocellulose, and immunoblotting were performed as previously described (10 (link), 30 (link)). After blocking, nitrocellulose membranes were incubated with human sera (1:50), rabbit IgG against the α3 chain of laminin 332 (1:10,000, Sigma Aldrich, Munich, Germany), monoclonal mouse IgG against the β3 and γ2 chains (clone A-6, 1:100,000; clone E-6, 1:10,000, respectively; both Santa Cruz Biotechnology, Dallas, TX, USA), diluted in TBS with 0.5% Tween-20 (TBS-T) containing 5% skimmed milk and 1% BSA. After washing with TBS-T twice for 12 min, the secondary antibodies, horseradish peroxidase (HRP)-conjugated monoclonal mouse anti-human IgG4 antibody (1:10,000, Southern Biotech, Birmingham, AL, USA), polyclonal rabbit anti-mouse IgG antibody (1:100,000, DAKO, Glostrup, Denmark), and polyclonal goat anti-rabbit antibody (1:10,000, DAKO) were used. After 1 h of incubation, an additional washing step with TBS-T was performed. The proteins were visualized using ECL prime detection systems (GE Healthcare Europe, Freiburg, Germany).

EGFP was detected by anti-GFP rabbit serum A-6455 (Life Technologies), ACTINB was detected by monoclonal anti-β-actin antibody (A5441, Sigma Aldrich), TUBA1A was detected by anti-alpha Tubulin antibody (ab80779, abcam), GAPDH was detected by Anti-GAPDH antibody (G4595, SIGMA Aldrich),PKR was detected by anti-PKR antibody (ab32052, abcam), p-PKR was detected by anti-PKR phospho T451 antibody (ab81303, abcam), eIF2-alpha was detected by anti EIF2S1 antibody (ab26197, abcam), p-eIF2-alpha was detected by anti-EIF2S1 phospho S51 antibody (ab32157, abcam), p-4E-BP1 was detected by anti-phospho-4E-BP1 (ser65) antibody (#9451, Cell Signaling Techonology) and 4E-BP1 was detected by anti-4E-BP1 antibody (#9452, Cell Signaling Technology). Secondary antibodies were horseradish peroxidase (HRP) conjugated Polyclonal Goat Anti-Rabbit antibody (P0448; Dako) and horseradish peroxidase (HRP) conjugated Polyclonal Goat Anti-Mouse antibody (P0447; Dako).