Laemmli sample buffer 4x concentrate

Each protein extract was adjusted to a 2 μg/μL concentration with the addition of Laemmli sample buffer (4X concentrate, Bio-Rad, Hercules, CA, USA), heated for 5 min at 95 °C, and separated on precast polyacrylamide gradient gels (7.5–15%). After transfer to 0.2 μm polyvinylidene difluoride membranes (PVDF, Bio-Rad), the membranes were blocked in either 5% (w/v) nonfat dried milk or 1% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich, Saint Louis, MO, USA) diluted in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBS-T), and probed overnight at 4 °C with the respective primary antibodies, as follows: mouse anti-actin (1:2500), rabbit anti-LDHA (1:1000), rabbit anti-microtubule-associated protein 1B/2B light chains 3B (MAP-LC3) (1:1000) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-HIF-1α (1:250), rabbit anti-PDK1 (1:1000), rabbit anti-Bax (1:1000), rabbit anti-Bcl-2 (1:500), and rabbit anti-caspase 3 (1:250) (Abcam, Cambridge, UK). Membranes were subsequently incubated with the respective HRP-linked anti-rabbit or anti-mouse IgG (1:1000) (Cell Signaling Technology, Danvers, MA, USA) antibodies and developed using ECL or ECL Plus (WesternBright Sirius Chemiluminescent Detection Kit, Advansta Inc., USA). Detection and densitometric quantification of band intensities was performed using a G-Box system (Syngene, Cambridge, UK).

For the Western blot analysis, protein levels in the whole-cell lysates were adjusted to a 2 μg/μL concentration. Next, Laemmli sample buffer (4x concentrate, Bio-Rad, Hercules, CA, USA) was added, and the samples were denatured (5 min at 95 °C). The samples were separated on 15% polyacrylamide gels and transferred to 0.2 μm polyvinylidene difluoride membranes (PVDF, Bio-Rad, Hercules, CA, USA). Next, the membranes were blocked in 1% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich, Saint Louis, MO, USA) solution in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBS-T). The membranes were further incubated with the primary antibodies: mouse anti-actin (1:2500) or rabbit anti-microtubule-associated protein 1B/2B light chains 3B (MAP-LC3) (1:1000) (Cell Signaling Technology, Danvers, MA, USA), and subsequently, with the HRP-linked secondary antibodies (anti-mouse or anti-rabbit IgG (1:1000) (Cell Signaling Technology, Danvers, MA, USA)). The reaction was developed using an ECL kit (WesternBright Sirius Chemiluminescent Detection Kit, Advansta Inc., USA). Bands detection and densitometric quantification were performed using the G-Box system (Syngene, Cambridge, UK).