Western ecl substrate

For further analysis of chondrogenic markers, the NSCs were lysed using RIPA lysis buffer (Sigma-Aldrich). A total of 30 µg protein was subject to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with the use of 10 % resolving gels, then followed by transfer to polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The membrane was incubated with primary antibodies specific for COL II (Abcam, ab34712), SOX9 (Santa Cruz, sc-166,505), Aggrecan (Santa Cruz, sc-70,332), COL I (Abcam, ab34710), and β-actin (Santa Cruz, sc-47,778), respectively, all of which were diluted in 1:1000. Those markers were visualized by chemiluminescence using Western ECL substrate (Thermo Scientific), and the luminescent images were analyzed using a LAS-3000 (Fujifilm).

Western blot analysis was performed according to published protocols [13] (link). The membrane was incubated at 4°C overnight with specific antibodies directed against STAT3 (1∶1000), pSTAT3 (1∶2000 diluted in distilled water with 5% BSA), Raptor (1∶500), p-Raptor (1∶500) or β-actin (1∶3000). After incubation with primary antibodies, the membrane was washed three times in TBS-0.1% Tween 20 for 15 min each before incubated with species specific secondary antibodies (1∶10000) for 1 h at room temperature. Secondary antibodies were diluted in TBS-0.1% Tween 20 with 5% milk if not differently indicated. To visualize the bands the membrane was incubated in clarity western ECL substrate (Thermo Fisher Scientific, Bonn, Germany) for 5 min for chemiluminescent detection and exposed to an ECL hyperfilm (Th. Geyer GmbH & Co. KG, Hamburg, Germany). For the analysis of β-actin, the membranes were stripped and reprobed with an anti β-actin antibody to account for protein loading variations. Relative density of bands was evaluated by densitometry with Image J software. The mean density of the untreated sample ( =  control) bands was assigned an arbitrary value of 1, and the mean density of the treated groups are expressed relative to the control groups.

Proteins were extracted from cell lysates following the manufacturer’s protocols (Beyotime, China). Protein concentration was quantified using the BCA protein assay kit (Thermo Fisher Scientific, United States) and 30 μg protein was separated in a 12% SDS polyacrylamide gel and electro transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, United States). Membranes were blocked with 5% (w/v) BSA for 2 h at room temperature and then incubated with primary antibodies with light shaking overnight at 4°C. Primary antibodies against UCP1, PGC-1α, TFAM, NRF1, CREB, P-CREB and GAPDH (abcam) were diluted to a ratio of 1:1,000 in TBST buffer. The membranes were washed 3 times for 5 min each with 10 mL of TBST [10 mM Tris-HCl, 150 mM NaCl and 0.1% (v/v) Tween-20] and then incubated with secondary antibody at room temperature for 2 h. Secondary antibodies goat anti-rabbit or goat anti-mouse (Proteintech, United States) were diluted to a ratio of 1:5,000 in TBST buffer. The membrane was incubated in Western ECL substrate (Thermo fisher or Proteintech, United States) and exposed to Tanon imager, using ImageJ software for image analyses.