Non fat dry milk tbs 0.1 tween

Lysates of mouse tissues or cells were homogenized in RIPA-Lysis buffer (150 mM NaCl, 50 mM TrisHCl pH 8, 5 mM EDTA pH 8, 5 mM EGTA pH 8, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS (Sigma-Aldrich) containing EDTA-free protease inhibitor cocktail (Roche Diagnostics) (1 tablet/50 ml). Cultured cells were homogenized by scraping and repeated pipetting, while tissues were disrupted using stainless steel beads and a Tissue-Lyser II (Qiagen). For analysis of secreted proteins, cell media were collected and concentrated using Amicon centrifugal filters. Supernatants were transferred to a new tube prior to determining the protein concentration using the bicinchoninic acid (BCA) kit (Sigma-Aldrich). After adding Laemmli buffer, lysates were boiled for 5 min at 95 °C and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto nitrocellulose membranes by electroblotting in a wet chamber (Bio-Rad Laboratories). The membranes were stained with Ponceau S and blocked with 5% non-fat dry milk TBS-0.1%Tween (Sigma-Aldrich). The blots were incubated with primary antibodies overnight at 4 °C and developed using horseradish peroxidase-conjugated secondary antibodies (Calbiochem) followed by chemi-luminescent detection with a Fujifilm analyzer (LAS-4000). Uncropped blots are found in the Source data file.

Cells and tissues (using the Tissue Lyser II, Qiagen) were homogenized with 3 volumes of RIPA lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS and 1 tablet cOmplete EDTA-free protease inhibitor cocktail (Roche) per 50 ml buffer), incubated for 10 min on ice and centrifuged for 10 min at 20,000g and 4 °C. Protein concentrations were determined using the Bicinchoninic Acid Assay (Sigma-Aldrich). Equal protein amounts were boiled in Laemmli buffer (1.7% SDS, 5% glycerol, 0.002% bromophenol blue, 60 mM Tris-HCl pH 6.8, 100 mM DTT) for 5 min at 95 °C, separated by SDS–PAGE and transferred onto nitrocellulose membranes by electroblotting in a wet chamber (Bio-Rad). The membranes were blocked for 1 h with 5% non-fat dry milk TBS-0.1% Tween (Sigma-Aldrich), incubated with the primary antibodies overnight at 4 °C, followed by three washes in TBS-0.1% Tween and incubation with a horseradish peroxidase-conjugated secondary antibodies (Calbiochem) for 2–3 h. Blots were then developed by chemiluminescent detection with a Fujifilm analyzer (LAS-4000) and signals quantified using ImageJ. Uncropped scans of all of the immunoblots are shown in Supplementary Figs 7 and 8.

Cells and tissues were homogenized (using the Tissue Lyser II, Qiagen) with 3 volumes of RIPA lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS and 1 tablet cOmplete^TM EDTA-free protease inhibitor cocktail (Roche)), incubated for 30 min on ice and centrifuged for 15 min at 16,000 × g and 4 °C. Protein concentrations were determined using the BCA assay (Sigma-Aldrich). Equal protein amounts were boiled in Laemmli buffer (1.7% SDS, 5% glycerol, 0.002% bromophenol blue, 60 mM Tris-HCl, pH 6.8, 100 mM DTT) for 5 min at 95 °C, separated by SDS–PAGE and transferred onto nitrocellulose membranes by electroblotting with the semi-dry Trans-Blot Turbo Transfer system (BioRad). The membranes were blocked for 1 h with 5% nonfat dry milk TBS-0.1% Tween (Sigma-Aldrich), incubated with the primary antibodies overnight at 4 °C, followed by three washes in TBS-0.1% Tween and incubation with a horseradish peroxidase-conjugated secondary antibodies (Calbiochem) for 2–3 h. Blots were then developed by chemiluminescent detection with a Fujifilm analyzer (LAS-4000) and signals quantified using ImageJ.