Sc 69879

Testicular tissue obtained from wild-type and [Dnmt3a^ADD/ADD, Dnmt3L^ADD/ADD] males of 10 weeks old was homogenized in ice-cold lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.5% Sodium Deoxycholate, 0.1% SDS, 1 mM EDTA, 1x protease inhibitor). Proteins were subjected to 8 or 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for 60 minutes at 100 V and transferred to a PVDF membrane. The membrane was blocked with skimmed milk for 30 minutes at room temperature and incubated overnight at 4 °C with primary antibodies against DNMT3A (NOVUS, 64B1446), DNMT3L (Abcam, ab194094) (dilution 1:1000), and β-actin (Santa Cruz sc-69879) (dilution 1:1000) in blocking buffer. Horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG antibodies (Abcam, ab6789 and ab6721) (dilution 1:30,000) were used as the secondary antibodies. The membrane was incubated with Chemi-Lumi One Ultra (Nacalai Tesque) and the resulting chemiluminescence signals were detected using an ImageQuant LAS3000 mini (Cytiva).

Protein expression levels were analyzed using western blot analysis. Briefly, total protein was harvested from the cells using RIPA lysis buffer (Beyotime, China), and protein concentrations were detected using a BCA kit (Thermo). After SDS-PAGE, the following primary antibodies against the target proteins were used: Erlin1 (1:1000, ab171372; Abcam), GAPDH (1:10,000, SC-32233; Santa Cruz Biotechnology), and β-actin (1:5000, SC-69879; Santa Cruz Biotechnology). GAPDH or β-actin was used as an endogenous control to normalize target protein expression [37 (link)].

Western blotting analysis was performed to detect the expression of P3H4 protein (SC65) in transfected A549 and NCI-H1299 cells. Cells were lysed with RIPA buffer (Beyotime Biotechnology, Shanghai, China). BCA Protein Assay Kit (Beyotime Biotechnology) was used to determine the total protein concentration. β-actin was used as an internal reference. A total of 50 μg of target protein was loaded onto 10% SDS-PAGE in different lanes, and then transferred to PVDF membrane. Subsequently, the PVDF membrane was blocked at room temperature for 1 h or overnight at 4 °C with blocking solution (TBST solution of 5% skimmed milk). The PVDF membrane was incubated with primary antibodies including anti-P3H4 (1:500); 15288-1-AP; Proteintech, Rosemont, IL, USA) and anti-β-actin (1:5000; sc-69879; Santa Cruz Biotechnology, Dallas, TX, USA) at room temperature for 2 h or overnight at 4 °C. The membrane was then incubated with a secondary antibody such as goat anti-rabbit IgG (HRP-linked goat anti-rabbit IgG, 1:10,000, # 7074, CST) or goat anti-mouse IgG (HRP-linked goat anti-mouse IgG, 1:10,000, # 7076, CST) for 1.5 h at room temperature. Finally, X-ray analysis was performed using 20× LumiGLO^® reagent and 20× Peroxide # 7003 kit (CST). The experiment was repeated three times.

For western blot analysis, the cells were lysed in RIPA buffer (50 mM TRIS pH 7.5; 0.1% Triton X, 1 mM EDTA, 135 mM NaCl) supplemented with Protease Inhibitor Cocktail (cOmplete™ ULTRA Tablets, EDTA-free, glass vials Protease Inhibitor Cocktail, Roche, Switzerland). Three replicates of isolated proteins were pooled together and 20 μg of proteins in total was separated on 10% polyacrylamide gels and transferred to nitrocellulose membranes (Amersham™Protran^® Premium 0.45μm NC; GE Healthcare, Chicago, IL, USA). The membranes were blocked for 1 h with TBS-T containing 5% milk (Milchpulver blotting grade, Roth, Karlsruhe, Germany) prior to incubation with primary antibodies. The primary antibodies used were diluted in blocking buffer as follows: 1:1000 anti-B4GALT1 (ab121362, Abcam, Cambridge, UK), anti-β actin 1:1000 (sc-69879, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Incubations were performed overnight at +4 °C. The membranes were then washed and incubated in HRP conjugated secondary goat anti-mouse or goat anti-rabbit antibodies (ab205719 and ab6721, respectively, Abcam) for 1 h at room temperature. The signals were developed using Immobilon Western Chemiluminescent HRP Substrate (Merck, Germany) and photographed using the Alliance Q9 Advanced imaging system (Uvitec, Cambridge, UK). The protein signals were quantified using ImageJ [46 (link)].

Hypertrophy was assessed by immunofluorescence for β-actin. Cells were fixed with PFA 4%, washed with PBS 1X and incubated overnight at 4°C with the primary antibody (sc-69879 Santa Cruz Biotechnology, Dallas, Texas). Afterword, cells were incubated with a FITC-conjugated secondary antibody for 1 hour at room temperature. Cell nuclei were stained with Hoechst (62249, Thermo Fisher). Images were acquired with a fluorescence microscope and cell size was quantified by ImageJ (National Institutes of Health, USA). Cell hypertrophy was also investigated by analyzing atrial natriuretic peptide (ANP) and β- heavy chain cardiac myosin (MHC) by RTqPCR and by western blot. Primers used for Nppa and β-Mhc were the following: β-Nppa For: 5’-TTCAAGAACCTGCTAGACCAC-3’, Rev: 5’-CCTCAGAGAGGGAGCTAAGT-3’ Mhc For: 5’-CCTCCCAAGTTCGACAAGAT-3’, Rev: 5’-AGGCCTGAGTAGGTGTAGAT-3’. Gapdh was used as houseeking gene.

For western blot, the glioma cells were lysed in lysis buffer (Thermo Scientific) supplemented with the protease inhibitor cocktails (B14012, Bimake) for 15 min. The supernatants were collected after centrifugation for 15 min at 12,000 rpm. After then, the 50 μg total proteins were loaded on SDS‐PAGE and transferred onto the PVDF membranes (Millipore). After blocking in 5% skimmed milk for about 1 h, the PVDF membranes were incubated with the indicated antibodies overnight at 4°C. The indicated primary antibodies were as follows: anti‐FHOD1 (ab206692, 1:1000, Abcam), anti‐HSPB1 (18284‐1‐AP, 1:1000, Proteintech), anti‐TRF1 (11899‐1‐AP, 1:1000, Proteintech) and anti‐β‐actin (Sc‐69,879, 1:5000, Santa Cruz). The protein levels were determined by the Immobilon Western Chemiluminescent HRP Substrates (Millipore).