Glyceraldehyde 3 phosphate dehydrogenase gapdh

Hypothalamic tissues and cell samples were dissociated in 200 μL of lysis buffer consisting of 1% phenylmethanesulfonyl fluoride (PMSF) (KGP610, KeyGEN, Nanjing, China), and the protein concentration was quantified by a BCA assay kit (Beyotime Biotech Inc., Shanghai, China). The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Roche, Mannheim, Germany). Five percent skim milk in Tris Buffered Saline Tween-20 (TBST) was used to block the membranes for 1 h at room temperature. Following this, the membranes were incubated with the following primary antibodies at 4 °C overnight: PSD95 (1:1000, abcam, Cat# ab12093, Cambridge, UK), Synaptophysin (1:1000, Proteintech, Chicago, IL, USA), FKBP51 (1:1000, Proteintech, Chicago, IL, USA), Lamin B1 (1:1000, Proteintech, Chicago, IL, USA), Progesterone Receptor (1:1000, Bioworld, St. Louis, MO, USA), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000, Proteintech, Chicago, IL, USA). After washing with TBST, the membranes were incubated with appropriate horse radish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. After being washed in TBST 4 times, protein bands were detected by enhanced chemiluminescence and Image J software (Version 1.43, NIH, Bethesda, MD, USA).

HDAC2 inhibitor CAY10683 was purchased from Sellect (Houston, USA). DMEM basic and fetal bovine serum (FBS) were obtained from Gibco (NY, USA). Lipopolysaccharide (LPS, purity of 99%), tumor necrosis factor alpha (TNF-α, purity of 97%), and D-galactosamine (D-gal, purity of 98%) were purchased from Sigma (St. Louis, USA). Rabbit anti-rat/human HDAC2, bcl2, cytochrome c (cyt c), apoptosis protease activating factor 1 (apaf1), caspase 3/cleaved-caspase 3, caspase 9, cleaved-caspase 9, histone H3 (H3), and acetylated histone H3 (AH3) antibodies were obtained from Cell Signaling Technology (Boston, USA). Rabbit anti-rat/human bax antibody was obtained from Abcam (Cambridge, UK). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cytochrome c oxidase subunit 4 isoform 1 (cox IV) antibodies were purchased from Proteintech (Wuhan, China). The goat anti-rabbit fluorescent secondary antibody (IRDye800) was obtained from LI-COR Biosciences Inc. (Lincoln, USA). RNAiso Plus, PrimeScript™ RT reagent, and SYBR Premix Ex Taq kits were purchased from TaKaRa (Dalian, China). The purified mitochondrial permeability transition pore (MPTP) fluorescence detection kit was purchased from GENMED (Shanghai, China).

These experiments were performed as previously described [15 (link)]. Briefly, 24 hours after transfection, the protein samples (separate membrane and cytosolic samples) were extracted from cells using the temperature-induced phase separation method. A total of 100 μg lysates or subcellular fractions were separated using 6 to 10% sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene fluoride (PVDF) membranes and probed with specific antibodies. The following antibodies were used: Ca_V1.2α1C (Alomone Labs, Jerusalem, Israel), ATP1A2 (Proteintech) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech). Signals of blots were collected through a Bio-RAD ChemiDox XRS detection system and analyzed by Quantity One analysis software (Bio-Rad, Hercules, CA, USA).

Total RNA was isolated from cells by using TRIzol reagent (Invitrogen, 15596026) according to the manufacturer’s instructions. For the reverse transcription reaction, cDNA was synthesized using 2 µg RNA with the PrimeScript RT Reagent Kit. RT-qPCR was performed using TB Green Premix Ex Taq II (Takara, RR820A) in a StepOnePlus real-time PCR instrument (Thermo Fisher Scientific). In this study, β-actin served as an internal control. The primer sequences used for RT-qPCR were as follows: FOSL1 (forward):
5′-GCCTGTGCTTGAACCTGA-3′, FOSL1 (reverse): 5′-TGCTGCTACTCTTGCGATG-3′; β-actin (forward): 5′-GATCATTGCTCCTCCTGAGC-3′, β-actin (reverse): 5′-ACTCCTGCTTGCTGATCCAC-3′. For Western blotting, the proteins of the cells were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, P0013B) according to the manufacturer’s instructions. Then, the proteins were separated by electrophoresis liquid and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% milk for 1 h, incubated with primary antibodies overnight, and then incubated with secondary antibodies for 1 h. The primary antibodies used in this study were FOSL1 (Cell Signaling Technology, #5281) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (Proteintech, 10494-1-AP).

Whole cell lysates were prepared by lysis buffer (50mM Tris-HCl, 150mM NaCl, 1% Triton-X 100, 1mM MgCl₂, MnCl₂ and CaCl₂, 1mM PMSF and 10mM sodium fluoride) [49 (link)]. The primary antibodies used included the following: DPT (HuaAn Biotechnology, Hangzhou, China), focal adhesion kinase (FAK) (Abcam, Cambridge, UK), p-FAK Tyr397 (Abcam, Cambridge, UK), Src (Cell Signaling Technology, Boston, MA), p-Src Tyr527 (Cell Signaling Technology, Boston, MA), ITGA3 (Abcam, Cambridge, UK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech Group, Chicago IL). After incubating with the IRDye 680 anti-mouse (LI-COR, Lincoln, NE) and IRDye 800 anti-rabbit (LI-COR, Lincoln, NE) secondary antibodies for 1 hour at room temperature, the bands were detected by an Odyssey infrared imaging system (LI-COR, Lincoln, NE). Quantification was analyzed by using Image J software.