Protein Expression Analysis of Rabbit Disc Tissues

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.

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Wang D., Hartman R., Han C., Zhou C.M., Couch B., Malkamaki M., Roginskaya V., Van Houten B., Mullett S.J., Wendell S.G., Jurczak M.J., Kang J., Lee J., Sowa G, & Vo N. (2021). Lactate oxidative phosphorylation by annulus fibrosus cells: evidence for lactate-dependent metabolic symbiosis in intervertebral discs. Arthritis Research & Therapy, 23, 145.

Publication 2021

proteinase inhibitor cocktail Ab150423 AB93048 Ab85319

Actin Anti antibody Dehydrogenase Female Ldha Lumbar Mct1 New zealand white rabbits Protein Proteinase inhibitor Pyruvate Rabbit T protein Tissue Tissue extracts Western blots

Corresponding Organization :

Other organizations : University of Pittsburgh, Brigham and Women's Hospital, Harvard University

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Variable analysis

independent variables

Tissue type (AF and NP)

dependent variables

Protein expression levels of hexokinase-1 (HK1)
Protein expression levels of MCT4
Protein expression levels of LDHA
Protein expression levels of MCT1
Protein expression levels of LDHB
Protein expression levels of pyruvate dehydrogenase (PDH)

control variables

Age of New Zealand White rabbits (6 months)
Sex of New Zealand White rabbits (female)
Extraction of tissue protein using T-PER Tissue Protein Extraction Reagent with proteinase inhibitor cocktail
Western blot protocol as described previously [22]
Loading control (β-actin)
Anti-rabbit HRP secondary antibody

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