Ab93048

The total protein from hypothalamus and colon were separated by SDS-PAGE and then transferred to PVDF membrane. The membrane was soaked with skimmed milk powder to reduce the binding of non-specific antibodies. The membranes were then exposed to primary antibodies and secondary antibodies (peroxidase-conjugated goat anti-rabbit IgG (111–035-003, Jackson) or peroxidase-conjugated goat anti-mice IgG (115–035-003, Jackson) depending on the primary antibody). Protein was quantified with Lab image Software (BioRad, USA) and expressed as relative units to housekeeping proteins.
The primary antibodies are as follows: anti-free fatty acid receptor 2 (FFAR2; ABC299, Merck Millipore), anti-monocarboxylic acid transporter 1 (MCT1, ab93048, Abcam), anti-small peptide transporters (PEPT1, ab203043, Abcam), anti-GAPDH (A01020, Abbkine), and anti-β-tubulin (A01030HRP, Abbkine).

AF and NP tissues were carefully isolated separately from lumbar discs of four 6-month-old female New Zealand White rabbits. Tissue protein extracts were prepared using T-PER Tissue Protein Extraction Reagent with proteinase inhibitor cocktail as per the manufacturers instructions (Cat. No 78510, Thermo Fisher). Western blots were performed as described previously [22 (link)] to detect hexokinase-1 (Anti-HK, Ab150423, Abcam), MCT4 (anti-MCT4, SC-376140, Santa Cruz Biotechnolgy), LDHA (anti-LDHA, PA5-27406, Invitrogen), MCT1 (anti-MCT1, AB93048, Abcam), LDHB (anti-LDHB, Ab85319, Abcam), and pyruvate dehydrogenase (PDH; anti-PDH, #2784S, Cell Signaling Technology). Loading control -actin (Cat. No. PA1-183, Thermo Fisher) and anti-rabbit HRP secondary antibody (Cat. No. 31460, Thermo Fisher) were used.

Adherent MDA-MB-231 and FaDu cells were lysed in RIPA buffer (Thermo Scientific, Merelbeke, Belgium) supplemented with 1% protease and phosphatase inhibitors (Thermo Scientific, Merelbeke, Belgium). Protein amount in whole-cell lysates was measured with a Pierce™ BCA Protein Assay Kit (Thermo Scientific, Merelbeke, Belgium). Equal amounts of protein were loaded onto 4–15% Mini-PROTEAN^®® TGX™ Precast Gels (Bio-Rad, Temse, Belgium). Following electrophoresis in 1x Tris/glycine/SDS running buffer (Bio-Rad, Temse, Belgium), proteins were transferred to PVDF membranes using the Trans-Blot^®® Turbo™ RTA Mini PVDF Transfer Kit (Bio-Rad, Temse, Belgium) according to the manufacturer’s instructions. Non-specific binding was blocked by soaking the membranes in 5% milk in tTBS (1x Tris-Buffered Saline, 0.1% Tween 20, Bio-Rad, Temse, Belgium) at room temperature for 1 h. Membranes were incubated with primary anti-HSP90 (4875S, Cell Signaling, Leiden, The Netherland), anti-MCT1 (Ab93048, Abcam, Amsterdam, The Netherland) and anti-MCT4 (AB3316P, Sigma-Aldrich, Hoeilaart, Belgium) antibodies in tTBS/milk 5% at 4 °C overnight, followed by incubation with anti-rabbit or anti-mouse secondary antibodies (Jackson IR, Ely, UK) in tTBS/milk 1% at room temperature for 1 h. Detection was performed using the SuperSignal™ West Pico Plus kit (Thermo Scientific, Merelbeke, Belgium).