Immobilon western chemiluminescent hrp substrate wbkls

Erythrocyte‐depleted BM cells were lysed with 1 × sodium dodecyl sulfate (SDS) buffer (P0015, Beyotime, Shanghai, China) and boiled at 100°C for 5 min. The cell extractions were resolved by SDS‐polyacrylamide gel electrophoresis (PAGE) and electrotransferred to polyvinylidene difluoride (PVDF) membranes (ISEQ00010, Sigma, Darmstadt, Germany), followed by overnight incubation with the primary antibodies at 4°C. Horseradish peroxidase‐conjugated anti‐rabbit antibody and anti‐mouse antibody (401353, 401253, Millipore, Burlington, Massachusetts, USA) were used as the secondary antibodies, and Immobilon Western Chemiluminescent HRP Substrate (WBKLS, Sigma) was used for the detection. A comprehensive list of antibodies for WB analysis is included in Supplementary Table S1.

For glandular tissues, young and aged labial salivary glands (n = 3 per group) were processed for protein extraction. The tissues were homogenated with 1× sodium dodecyl sulfate (SDS) buffer (P0015, Beyotime, Shanghai, China) and boiled at 100°C for 5 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene difluoride(PVDF) membranes (ISEQ00010, Sigma, Darmstadt, Germany), followed by overnight incubation with the primary antibodies (anti-IL-6 and anti-TNF-α at 1:1000 dilution) at 4°C. Active bands were detected using conjugated horseradish peroxidase-conjugated anti-rabbit antibody and anti-mouse antibody (401353,401253, Millipore, Burlington, Massachusetts, USA). Detection was performed by Immobilon Western Chemiluminescent HRP Substrate (WBKLS, Sigma).