Ab3242

For western blot analysis, proteins collected from whole lung tissue were separated by SDS-PAGE in a 15% polyacrylamide gel for 3 h. The separated proteins were then transferred to PVDF membranes (Bio-Rad, USA) for 1 h at 90 volts. After blocking the membranes overnight at 4 °C with 5% skim milk, they were probed using anti-GAPDH (Santa Cruz Biotechnology, LF-PA0018), polyclonal mouse anti-NF-κB (Santa Cruz Biotechnology, SC-71675), monoclonal mouse anti-TNF-α (ABfrontier, AB1793), polyclonal mouse anti-IL-1β (ABfrontier, AB1413), polyclonal mouse anti-Cathelicidin (ABfrontier, AB93357) and polyclonal mouse anti-PGC-1 (Millipore AB3242). The membranes were then washed with TBST buffer and incubated with secondary antibodies (goat anti-mouse IgG (HRP) LF-SA5001-conjugated). Finally, the blots were developed using a western blot detection kit (LF-QC0103, Abfrontier)74 (link).

For each sample, the muscle cross-section that was cut immediately following those used for MHC immunolabeling was used to determine PGC-1α content in situ. Sections were first allowed to reach room and were fixed in acetone at 4°C for 15 min. Samples were then washed for 5 min in PBS (pH 7.4) at 4°C before being incubated for 15 min in a permeabilization solution 0.1% Triton X-100 in PBS) at 4°C. Slides were then washed 3 times in PBS, before being incubated at 4°C in a blocking solution (10% goat serum in PBS) for 15 min at room temperature. Slides were then incubated overnight at 4°C with a rabbit IgG polyclonal anti- PGC-1α (Millipore, Ab3242, 1∶50). For mouse muscle cross-sections a mouse IgG monoclonal anti-dystrophin (Sigma, D8168, 1∶100) was also applied. The following day, slides were washed 3 times in PBS at 4°C before being incubated for 90 minutes at room temperature with an Alexa Fluor 488 IgG goat anti-rabbit (A-11008, 1∶500) and an Alexa Fluor 647 goat anti-mouse IgG, (Invitrogen, A-21235, 1∶100) for mouse muscle cross-sections. Cross-sections were washed 3 times in PBS at 4°C before a 10 min incubation in a PBS solution containing DAPI (Invitrogen, D1306, [300 nm]) at 4°C. Slides were then washed 3 times in PBS and finally cover slipped using Prolong Gold (Invitrogen, P36930) as mounting medium.

Gastrocnemius and hearts lysates (obtained by centrifugation of homogenates) were separated by SDS-PAGE, transferred to nitrocellulose membranes and probed using antibodies against MyoD, Myogenin (M3512, M3559-Dako), Desmin, Collagen type-III, α-SMA (D1033, C7805, A5228-Sigma-Aldrich), PGC1α, β-ATPase (AB3242, MAB3494-Millipore), TFAM, Tom20, CSQ2, fsTn-I, VEGF, VE-Cadherin, PECAM, SDHA and PPARγ (sc-23588, sc-11415, sc-390999, sc-377382, sc-7269, sc-9989, sc-46694, sc-377302, sc-7273-Santa Cruz Biotechnology), CoxIV (Ab14744-Abcam) and the appropriate secondary antibodies. α-tubulin or actin (T5168, A3853-Sigma-Aldrich) were used for normalization. Quantification was performed as previously described (Ferraro et al., 2016 (link)).

100.000 HEK or N2A cells were disseminated on polylysine coated coverglasses and incubated in DMEM over night. Cells were fixed with paraformaldehyde for 15 minutes and blocked with BSA 5% for 1h at room temperature. 1. Antibodies (AB) were incubated over night: IgG 1:200, PGC-1α Millipore AB3242 1:200, nucleolin 1:200. Second AB used for immunofluorescence: alexa 555 or 488 1:200 with incubation-time of 45 minutes. Staining of nuclei was performed with DAPI 1:5000.

Monoclonal antibodies against the TFIIH subunits, RNA pol II, GST-Tag and Flag-Tag were produced at the IGBMC. Antibodies against PEPCK (sc-74823, Santa Cruz SC), G6Pase (Ab83690, AB Cam LTD), PGC-1α (4C1.3, Calbiochem; AB3242, Millipore), SIRT1 (2028, Cell Signaling Technology CST and 3H10.2 Millipore, for murine and human SIRT1, respectively), HNF-4α (C-19, SC), FOXO1 (L27, CST), acetylated FOXO1 (D-19, SC), CREB (48H2, CST), β-tubulin (KMX-1, Millipore) and acetylated lysine (9441, SCT) were purchased.