Odyssey fc device

Purified lung epithelial cells were lysed in 2x Western blotting buffer (120 mM Tris-Cl pH 6.8, 4% SDS, 20% Glycerol, 200 mM DTT, 0.02% Bromophenol Blue). The lysates were centrifuged at 16,100 x g for 15 min at 4 °C before loading onto SDS-PAGE. The primary antibodies used were rabbit anti-Raptor (1:2000, EMD Millipore Corporation, Cat# 09-217; RRID:AB_1659713), mouse anti-S6 ribosomal protein (1:2000, Cell Signaling Technology, Cat# 2317; RRID:AB_2238583), rabbit anti-phospho-S6 ribosomal protein (Ser235/236) (1:2000, Cell Signaling Technology, Cat# 4856), rabbit anti-MLC2 (1:500; Cell Signaling Technology, Cat#3672; RRID:AB_10692513), goat anti-TFAM (1: 250, Santa Cruz Biotechnology, Cat# sc-23588; RRID:AB_2303230), rabbit anti-COX10 (1:500, Proteintech, Cat# 10611-2-AP; RRID:AB_2084833), mouse anti-MTCO1 (1:500, Abcam, Cat# ab14705; RRID:AB_2084810), and mouse anti-alpha-tubulin (1:3000, Developmental Studies Hybridoma Bank, Cat# 12G10; RRID:AB_1157911). Western blotting images were captured on a LI-COR Odyssey Fc device. The uncropped images are available in the Source Data file.

Proteins were separated by 8% or 10% SDS–PAGE and transferred onto PVDF membranes (0.45 mM, #T830.1; Carl Roth). The membranes were blocked in 5% skimmed milk powder (Sigma-Aldrich) or in casein solution (Sigma-Aldrich) for 1 h at RT. Primary antibodies were incubated overnight at 4°C in 5% skimmed milk and 0.1% Tween. Membranes were washed 3× in TBS/0.05% Tween and incubated with secondary antibody for 1 h at RT. Membranes were washed 3× in TBS/0.05% Tween. Proteins were detected either after short incubation in Immobilon Western ECL substrate (#WBKLS0500; Millipore) on an Odyssey FC device (Li-cor Biosciences) or on an Odyssey CLx scanner (Li-cor Biosciences). The following antibodies were used: ADAM19_74–123 (#NBP1-69367; Novus), ADAM19_218–267 (#ab104800; Abcam), FLAG (#TA50011-100; Origin), PTHR1_4–54 (#ABIN2776779; Antibodies-Online.de), PTHR1_52–86 (#ABIN5708269; Antibodies-Online.de), FLAG (#TA50011-100; Origin), HA (#137838; Abcam), polyclonal PTHR1_573–593 (Lupp et al, 2010 (link)), anti-mouse IgG-HRP (#P0260; Dako), anti-rabbit IrDye680 (#926-32223; Li-cor Biosciences).

The detailed methods were described in previous study [38 (link)]. Brain tissue and treated cells were homogenized in cold cell lysis buffer (9803S, Cell Signaling Technology) containing protease inhibitors and phosphatase inhibitors, then subjected to protein extraction. Protein samples were heated at 95 °C for 5 min, then mixed with loading buffer at a 1:1 ratio. Samples were subjected to electrophoresis on 8% or 12% gels (SDS-PAGE Gel Kit, P1200, Slaribio; 30 μg total protein per well) and transferred to a nitrocellulose membrane (66,485, BioTrace) at 4 °C for approximately 1–2 h (transfer time was dependent on protein weight). Subsequently, the membrane was incubated at room temperature for 1 h with 5% milk, then incubated overnight at 4 °C with primary antibody (listed in Additional file 1: Supplementary Table S2). The membrane was washed four times (5 min each) with Tris-buffered saline plus 0.1% Tween (TBST, T1085, Slaribio), then incubated at room temperature for 1 h with secondary antibody (listed in Additional file 1: Supplementary Table S2). Finally, the membrane was washed with TBST buffer and protein signals were quantified using an Odyssey Fc device (LI-COR Biosciences, Lincoln, NE, USA).