Anti rabbit hrp secondary antibody

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.

Ten tail discs from each mouse were used to assess aggrecan fragmentation utilizing a previously established method using anti‐aggrecan primary antibody (Cat. No. ab36861, Abcam) and anti‐rabbit HRP secondary antibody (Cat. No. 31460, Thermo Fisher; Ngo, Pohl et al., 2017). Expression of p53 protein was examined by extracting protein from spine discs using T‐PER Tissue Protein Extraction Reagent with proteinase inhibitor cocktail as per the manufacturer's instructions (Cat. No 78510, Thermo Fisher) and running a Western blot using the p53 (Cat. No. 2524, CST) and β‐actin (Cat. No. PA1‐183, Thermo Fisher) primary antibody and anti‐rabbit HRP secondary antibody (Cat. No. 31460, Thermo Fisher).

Cell lysates from Raw264.7 cells infected with CPER-derived or WT MNVs or Mock were harvested in KALB lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% (v/v) Triton X-100, 1 mM EDTA) supplemented with protease inhibitor cocktail III (Austral Scientific). Cell lysates from Raw264.7 cells infected with CPER-derived or WT MNVs or Mock were harvested in KALB lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% (v/v) Triton X-100, 1 mM EDTA) supplemented with protease inhibitor cocktail III (Austral Scientific). Samples were separated on a polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% BSA/TBS-T for 2 h at room temperature prior to probing with primary antibodies overnight at 4 °C. Primary antibodies, anti-MNV1 capsid antibody 5C4.10 (Cat: MABF2097 Merck Millipore) or anti-actin antibody (Cat: A2066 Sigma), were each diluted 1:1000 in 5% BSA/TBS-T. The following day, the membrane was washed three times with TBS-T and incubated with either anti-mouse HRP secondary antibody (Cat: G-21040, Thermo Fisher Scientific) or anti-rabbit HRP secondary antibody (Cat: A16035, Thermo Fisher Scientific). Secondary antibodies were prepared by 1:10,000 dilution in TBS-T. Probed membranes were developed with the Amersham ECL Western Blotting Detection Reagent and imaged on the GE Healthcare Life Sciences AI600 Imager.