Anti gpr27 pa5 110977

Protein expression levels were assessed through Western blot analysis. Proteins were extracted from the U87 and U251 cell lines utilizing RIPA lysis buffer, and their concentrations were subsequently semi-quantified using a BCA protein assay kit provided by Santa Cruz Biotechnology (Dallas, TX, USA). Proteins, in the amount of 20 µg per lane, were resolved via 12% SDS-PAGE and subsequently transferred onto PVDF membranes. These membranes were then blocked using 5% BSA sourced from Sigma-Aldrich for 1 h at ambient temperature before the blocking solution was discarded. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies: anti-GPR27 (PA5-110977, Invitrogen, Cambridge, MA, USA) and anti-GAPDH (MA1-16757; Invitrogen, Cambridge, MA, USA), both diluted at a ratio of 1:2000. Following the primary antibody incubation, the membranes were washed with TBST containing 0.1% Tween. Then, an HRP-conjugated secondary antibody was applied to incubate at room temperature for 1 h. Detection of the proteins of interest was achieved using the ECL Western Blotting substrate kit (Cattegory #ab65623; Abcam). Each experiment was repeated three times independently.