Iseq00010

Collecting cells and using the radio immunoprecipitation assay (RIPA) buffer (P0013B, Beyotime, Shanghai, China) in the presence of a phenylmethyl sulfonylfluoride (#8553, CST). Protein concentration were determined by the BCA Protein Assay Kit (#P0009, Beyotime, China). Equivalent amounts of the proteins (40 μg/line) were separated on Tris-Tricine Ready Gel (#1611210, Bio-Rad, Hercules, Calif, USA) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for nitrocellulose membrane polyvinylidene fluoride (PVDF) (ISEQ00010, Merck Millipore, Massachusetts, USA) blotting. The blotted membranes were blocked with 5% skim milk (#P0216-300g, Beyotime, Shanghai, China) for 1 hour at ambient temperature and incubated with primary antibodies overnight at 4^◦C. They were washed with tris-buffered saline Tween-20 (TBST) (CW0043S, CWBIO, Jiangsu, China), and the immunoreactive bands were visualized by enhanced chemiluminescence reagent. All bands were analyzed by NIH image 1.62. All experiments were conducted in triplicate.

Proteins were extracted from the cells at the indicated times using SDS lysis buffer (P0013G, Beyotime, China). Nuclear and Cytoplasmic Extraction Reagents (P0028, Beyotime, China) were used to separate the cytoplasmic and nuclear extracts from cultured cells. The protein samples (30 μg) were subjected to 4–20% polyacrylamide gels (JC-PE022/JC-PE022R, Genshare biological, China) to be electrophoresed and transferred to 0.22 μm PVDF membranes (ISEQ00010, Merck Millipore, USA) at 90 V for 1.5 h. Membranes were blocked with 5% skim milk for about 1 h at room temperature and then incubated with primary antibodies against HPV 16 E6 (sc-460, Santa Cruz, USA; 1:200), IκBα (4814, CST, USA; 1:1000), p-IκBα (Ser 32/36) (9246, CST, USA; 1:1000), p65 (10745-1-AP, Proteintech, USA; 1:1000), p-p65 (Ser536) (3031, CST, USA; 1:1000), p-p65 (Ser468) (3039, CST, USA; 1:1000), H3 histone (4499, CST, USA; 1:1000), Akt (10176-2-AP, Proteintech, USA; 1:1000), p-Akt (Ser473) (4060, CST, USA; 1:1000), p-Akt (Thr308) (13038, CST, USA; 1:1000), GAPDH (Proteintech, USA; 1:10,000), and β-actin (3700, CST, USA; 1:1000) at 4 °C overnight. Thereafter, the membranes were probed with IR Dye-labeled secondary antibodies (800CW, LI-COR Bioscience, USA; 1:10,000) and the signals were observed using an Odyssey Infrared Imaging System (LI-COR Bioscience, USA).

Cells were lysed in RIPA buffer with 2% proteinase inhibitor. 30 μg of total proteins were resolved on 4–20% precast polyacrylamide gels (4561096, Bio-Rad, Hercules, CA, USA), and transferred to PVDF membranes (ISEQ. 00010, Merck Millipore, Darmstadt, Germany). After transfer, the membranes were blocked with 5% Blotting-grade Blocker (1706404, Bio-Rad, Hercules, CA, USA) in PBS-T for 1 hour, and incubated with specific primary antibodies in the blocking solutions overnight at 4 °C. The membranes were then washed with PBST three times and incubated with secondary antibodies for 1 hour at room temperature. Then, the antibodies were detected using an ECL HRP substrate system (K-12045-D20, Advansta, Menlo Park, CA, USA). The protein band intensities were quantified using Image Lab software. Information regarding the antibodies is shown in the Supplemental Information.

Cells were then lysed in RIPA buffer (P0013B, Beyotime, Shanghai, China) containing a protease cocktail inhibitor (4693132001, Roche, Shanghai, China). We analyzed proteins using a BCA assay kit (P0009, Beyotime Institute of Biotechnology). After separating the proteins by SDS-PAGE, we next transferred the embedded ones onto PVDF membranes (ISEQ00010, EMD Millipore, Shagnhai, China). Subsequently, the membrane was blocked with 5% non-fat milk in TBS buffer at room temperature (RT) for 2 h and overnight incubation was undertaken with the following primary antibodies: anti-MORC2 (1: 1000), anti-HDAC4 (1: 1000), anti-p53 (1: 1000), anti-p21 (1: 1000), and anti-β-actin (1: 5000). The membranes were incubated with the corresponding IRDye^® 800CW Goat anti-Rabbit IgG (H + L) (1: 20,000; 926-32211; LI-COR) at RT for 1 h after three washes with TBST and we then imaged the blot using the LI-COR Odyssey^® CLx imager.

Western blotting was conducted as described previously with some modifications [27 (link)]. Briefly, cells were lysed with an appropriate amount of RIPA lysis buffer (Beyotime, P0013B) on ice and centrifuged at 13,000 rpm for 15 min at 4 °C. The supernatants were collected and processed with 5 × SDS-PAGE loading buffer. The protein concentration of all samples was measured using the BCA assay (Thermo Scientific, 23,227). Protein samples (about 30 μg) were separated on 10% SDS-gel electrophoresis and transferred to PVDF membranes (Merck Millipore, ISEQ00010). Then, the membranes were incubated with the primary antibody overnight at 4 °C, followed by Alexa Fluor-conjugated secondary antibodies. Detection was performed using the Odyssey Infrared Imaging System (LI-COR, Biosciences, Lincoln, NE, USA).

The cultured cells were collected and lysed in RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). Equal amounts of protein were separated by SDS-PAGE electrophoresis and transferred to the PVDF membrane (#ISEQ00010, Merck Millipore, Burlington, MA, USA). The following antibodies were used for Western blot analysis: mouse antibodies-PTH (1:500, #sc-69930, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CaSR (1:2000, #MA1-934, Invitrogen Waltham, MA, USA), and GAPDH (1:3000, #LF-PA0212, AbFrontier, Seoul, Korea).

The hippocampal tissue was placed in the RIPA lysate with PMSF protease inhibitor for homogenization, followed by centrifugation at 12,000 rpm at 4 ℃ to collect the supernatant. A Bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, P0010, Shanghai, China) was used for measuring the protein concentration, and the proteins were subsequently treated with RIPA lysis buffer for further processing. The sample was then boiled for 10 min, cooled and subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) for separation. The protein that has been isolated was moved onto a polyvinylidene fluoride (PVDF) membrane from Merck Millipore (ISEQ00010, USA). After an hour of using 5% skim milk to block the PVDF membrane. And then it was exposed to primary antibodies: SIRT1 (1:1000, 9475S, Cell Signaling Technology), BDNF (1:1000, ab108319, Abcam), and GAPDH (1:1000, ET1601-4, HUABIO) overnight in cold storage at 4 ℃. The membranes were then treated with an antibody conjugate with horseradish peroxidase (1:2000, Beyotime) for an hour at room temperature. The protein bands were then subjected to a quantitative analysis utilizing ImageJ software and an Enhanced Chemiluminescence (ECL) detection system (Beyotime).