Gapdh

The following antibodies were used: mtIF2 (1:200, Santa Cruz Biotechnology, Dalla, TX, USA, sc‐365477); mtIF3 (1:2000, Origene, Rockville, MD, USA, TA800421); mtEFTu (1:5000, Abcam, Cambridge, UK, ab173300); mtEFTs (1:10,000, Abcam, ab173528); mtEFG1(1:3000, Abcam, ab173529); mtRF1L (1:1000, Proteintech, Rosemont, IL, 16694‐1‐AP); mtRRF1 (1:1000, Proteintech, 12357‐2‐AP); mtEFG2 (1:1000, Proteintech, 16941‐1‐AP); TACO1 (1:3000, Proteintech, 21147‐1‐AP); insulin receptor substrate 1 (IRS‐1, 1:3000, Cell Signaling Technology, Danvers, MA, USA, #3194); hormone sensitive lipase (HSL, 1:3000, Cell Signaling Technology, #18381); glyceraldehyde 3‐phosphate dehydrogenase (GAPDH, 1:10000, Wako, Osaka, Japan, 016‐25523); β‐tubulin (1:10,000, Wako, 014‐25041).
We used the Total OXPHOS Rodent WB Antibody Cocktail (1:3000, Abcam, ab110413) to detect the proteins involved in mitochondrial oxidative phosphorylation (OXPHOS). This cocktail contains five monoclonal antibodies: mitochondrial NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8 (NDUFB8, ab110242); mitochondrial succinate dehydrogenase [ubiquinone] iron‐sulfur subunit (SDHB, ab14714); mitochondrial cytochrome b‐c1 complex subunit 2 (UQCRC2, ab14745); mitochondrial encoded cytochrome c oxidase I (MTCO1, ab14705); mitochondrial ATP synthase subunit alpha (ATP5A, ab14748).

Harvested cells were lysed in RIPA buffer (Thermo Fisher Scientific), and equal amounts of lysate proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA) using a Trans-Blot Turbo (Bio-Rad). After blocking with Bullet Blocking One (Nacalai Tesque, Kyoto, Japan), the membranes were incubated with primary antibodies against A1AT (1:2000; Dako, Tokyo, Japan), ERK1/2 (1:2000, #4695; Cell Signaling Technology [CST], Tokyo, Japan), phosphorylated (p-) ERK1/2 (1:2000, #4370; CST), p-38MAPK (1:2000, #8690; CST), p-p38MAPK (1:2000, #4511; CST), JNK (1:2000, #9252; CST), p-JNK (1:2000, #9251; CST), NFkB (1:2000, #4764; CST), IkB (1:2000, #4814; CST), p-IkB (1:2000, #2859; CST), CREB (1:2000, #9197; CST), p-CREB (1:2000, #9198; CST) hCGB (Thermo Fisher Scientific), or GAPDH (1:5000, 5A12; Fujifilm Wako Pure Chemical Corp.). After washing, the membranes were incubated with horseradish peroxidase-linked goat anti-rabbit or anti-mouse IgG (1:5000; Vector Laboratories, Burlingame, CA, USA). Immunoreactivity was detected with a chemiluminescence (Merck Millipore, Burlington, MA, USA) [7 (link)].

Immunoblotting with Immunostar LD chemiluminescent substrates (290–69904, Wako) was performed. Signals were detected with an LAS-3000 image analyser (Fujifilm, Tokyo, Japan) as previously described^{49 (link),50 (link)}. Lamin β1 (LMNB1; PM064, 1:10000) was purchased from MBL (Nagoya, Japan). CTSB (ab58802, 1:400), CIDEC/FSP27 (ab16760, 1:2000), PLIN1 (ab61682, 1:400) and PLIN2 (ab108323, 1:1000) were purchased from Abcam (Cambridge, UK). FLAG M2 (F1804, 1:5000) was purchased from Sigma-Aldrich. PPARG (sc-7273, 1:2000) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (010-25521, 1:5000) was purchased from Wako.

The cells were harvested in a sodium dodecyl sulfate (SDS) buffer (1 mM trisaminomethane-hydrochloride [pH 7.4], 2% SDS, 150 mM sodium chloride [NaCl], and 1 mM ethylenediaminetetraacetic acid) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). The lysates were then incubated at 95 °C for 10 min. Protein concentration of each lysate was examined using the DC protein assay kit (Bio-Rad, Hercules, CA, USA). An aliquot of a sample (20 µg) was subjected to SDS–polyacrylamide gel electrophoresis. The gel was transferred to an Immobilon-P polyvinylidene fluoride membrane (Merck Millipore, Burlington, MA, USA) and stained with antibodies against TAK1, phospho-JNK, JNK, phospho-p38 (P-p38), p38, phospho-ERK, ERK, phospho-p65 (Cell Signaling Technology), ASK1, GAPDH, and Iba1 (FUJIFILM Wako). NeuN antibodies were obtained from Abcam (Cambridge, UK). Uncropped data of Figs. 3, 4, 5 and 6 are shown in supplemental data (Supplemental Fig. 8–11).