Beta actin β actin

Immunoblotting technique was performed as we’ve previously described^{102 (link)}. Briefly, one million (1.0 × 10⁶) serum-containing or serum-deprived cells were stimulated with 250 μM H₂O₂ for 1 h or TNFα (0.1 ng/mL) for 30 min. Alternatively, cells were pre-treated with 5 mM NAC for 1 h followed by 250 μM H₂O₂ for 1 h. Cells were lysed and sonicated in 1 × Cell Signaling Technology lysis buffer prior to incubation on ice for 30 min. Lysates were centrifuged at max speed for 10 min at 4 °C, and then equal amounts of protein per sample were separated by SDS-PAGE and transferred to PVDF membrane. Protein bound membranes were blocked in 5% BSA/1XTBST and subsequently incubated with primary antibodies p27^Kip1 (1:1,000, Cell Signaling Technology) or pRB (1:1,000, Cell Signaling Technology) overnight at 4 °C in 5% BSA/TBST. Beta actin (β-actin; 1:1,000; Santa Cruz Biotechnology) served as a loading control. Primary antibodies were detected by HRP-conjugated secondary antibodies (1:10,000, Jackson ImmunoResearch) diluted in 5% BSA/1 × TBST. Protein expression was detected with chemiluminescence (Luminata Western HRP Chemiluminescence Substrates; Millipore Sigma) on ChemiDoc MP Imaging System (Bio-Rad, USA).

The following purified antibodies were purchased: hypocretin 1 (Santa Cruz Biotechnology sc‐80263, 1:250), hypocretin 2 (Abcam, ab229714, 1:500), beta‐actin (β‐actin) (Santa Cruz Biotechnology, SC‐8432, 1:1000), prepro‐hypocretin (Millipore and Sigma, AB3096, 1:500), P38 (Cell Signaling, #9212, 1:1000) and phospho‐p38 (Cell Signaling, #4511, 1:1000). Recombinant tumour necrosis factor alpha (TNF‐α) was obtained from R&D Systems. TNF‐α neutralizing antibody was purchased from BD Biosciences and Cell Signaling (11 969, used as indicated). Rat IgG was purchased from Jackson ImmunoResearch (415 005 166, used as indicated). The hypocretin (orexin) receptor 1 (HcrtR1/OX1R) antagonist SB334867 was purchased from Tocris (United Kingdom). All other reagents were purchased from Cell Signaling, Thermo Fisher Scientific (P‐SUWANEE, GA), Sigma‐Aldrich or Bio‐Rad.