Aifm1

pCMV6-Alox5-GFP (Origene) was transfected into 293T cells with vectors expressing FLAG-tagged MAPKs. The cells were lysed 24 h later and immunoprecipitated with anti-GFP (Origene), followed by Western blot with anti-phospho-Alox5^S217 (Cell Signaling Technology, Danvers, MA, USA). The blots were also probed with anti-GFP. Total lysates were used for Western blot by probing with anti-FLAG (Sigma, St. Louis, MO, USA). JMR cells transfected with siRNA targeting specific genes or non-targeting siRNA control were lysed for Western blot by probing with specific antibodies. The following antibodies were used for Western blot: rabbit polyclonal or monoclonal antibodies to Alox5, ERK1, p38 MAPK, p44/42 MAPK (ERK1/2), phosphor-p44/p42 MAPK (p-ERK1/ERK2), RAD51, SAPK/JNK, SESN2 (Cell Signaling Technology), Alox5AP, DHODH, HADHA, MGST1, TIA-1 (Abcam), EndoG (ProSci), OXR1 (Bethyl Laboratory, Montgomery, TX, USA.) and PDP1 (Sigma); and mouse monoclonal antibodies to AIFM1, API5, PNKP (Santa Cruz Biotechnology), caspase-3, caspase-6 caspase-7, caspse-9, phosphor-p38 MAPK, phosphor-SPAK/JNK (Cell Signaling Technology), FLAG (Sigma) and GFP (Origene). The blots were also probed with mouse monoclonal anti-α tubulin (Santa Cruz Biotechnology) to ensure equal loading.

Protein preparation and immunoblotting methods were described in previous studies^{5 (link)}. The following antibodies were used: aggrecan (Millipore Sigma, AB1031, 1:1000), MMP13 (Abcam, ab39012, 1:1000), SIRT5 (Proteintech, 15122-1-AP, 1:500), TOM20 (Proteintech, 11802-1-AP, 1:1000), AIFM1 (Santa Cruz Biotechnology, sc-13116, 1:500), CHCHD4 (Proteintech, 21090-1-AP, 1:500), NDUFB8 (Complex I) (Abcam, ab192878, 1:2000), SDHB (Complex II) (Abcam, ab175225, 1:2000), UQCRC2 (Complex III) (Absin, abs116449, 1:2000), COX IV (Complex IV) (Cell Signaling Technology, 4844s, 1:2000), ATP5F1A (Complex V) (Abcam, ab176569, 1:2000), and succinylated lysine (PTM Bio, PTM-419, 1:500).