Ab150423

Sperm proteins involved in fat and glucose metabolic pathways were selected for validation using western blot. Three key proteins hexokinase-1 (HK1), ATP citrate lyase (ACLY) and fatty acid synthase (FASN) were applied on individual samples obtained from RPL patients (n = 10) and the control group (n = 7). Proteins from each individual sample of the control and RPL sperm were used for western blot analysis. Primary antibodies: anti-β-actin mouse antibody (Sigma, A5441), anti-HK1 rabbit polyclonal antibody (Abcam, ab150423), anti-ACLY rabbit polyclonal antibody (Abcam, ab40793) and anti-FASN rabbit monoclonal antibody (Abcam, ab128856). Bands were revealed using a chemiluminescence reagent (ECL kit, PerkinElmer, Boston, MA, USA).

Cells were grown on glass coverslips coated with poly-D-lysine and laminin and fixed with ice-cold 4% paraformaldehyde in PBS for 15 min. Next, cells were permeabilized with 0.1% v/v Triton X-100 in PBS for 10 min, and then blocked with blocking buffer (10% FBS in PBS with 0.1% v/v Triton X-100) for 1 h. Blocking buffer was also used to dilute primary and secondary antibodies. The primary antibodies used were HK1 (LS-B6098, mouse monoclonal, 1:1000), HK1 (Abcam ab150423, rabbit monoclonal, 1:1000), GFAP (Abcam ab53554, goat polyclonal, 1:1000), and were incubated with the cells for 1 h at RT or overnight at 4 °C. The secondary antibodies (Alexa Fluor 488 donkey anti-mouse IgG, 1:500; Alexa Fluor 568 donkey anti-rabbit IgG, 1:500; Alexa Fluor 647 donkey anti-goat IgG, 1:500) were incubated with cells for 1 h at RT.

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.