Western Blot Analysis of Cellular Proteins

Cells (0.4–0.6 × 10⁶) were lysed in 25 μl SDS lysis buffer, and cell lysates were resolved by 10% SDS–polyacrylamide gel electrophoresis followed by Western blot analysis as described earlier (Saini et al, 2022 (link)). Primary antibodies were used at the following dilution: Notch1 (1:500), SLC43A2 (1:500), SLC7A5 (1:500), actin (1:1,000), HES1 (1:500), and tubulin (1:1,000) diluted in 5% skimmed milk in Tris-buffered saline–Tween 20 (TBST). Horseradish peroxidase–conjugated secondary antibody was used at a 1:1,000 dilution. The membranes were developed using Super Signal West Dura substrate (Thermo Fisher Scientific), and images were acquired in iBright FL1000 (Invitrogen).