Monoclonal anti gapdh antibody

Total protein samples were extracted and the concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Sample lysates (10 μg of protein) were separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was incubated with specific antibodies for SLC7A11 (1:1,000) or cyclin D1 (1:3,000; Abcam, Cambridge, MA, USA) at 4°C overnight followed by incubation with secondary antibodies. Protein levels were normalized to those of total GAPDH using a monoclonal anti-GAPDH antibody (Sigma-Aldrich Corporation, St. Louis, MO, USA). Autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad).

Western blots were performed as previously described [2 (link)]. In brief, 10 μg protein of each sample was separated by SDS-polyacrylamide gel electrophoresis using 12% gels. The resolved proteins were transferred to PVDF membranes (Millipore) at 300 mA for 45 min or 1 h. The membranes were blocked for 1 h with Tris-buffered saline-Tween 20 (TBST) containing 5% BSA at room temperature with slow shaking. The blocked membranes were probed with polyclonal anti-PDE3A antibody (Santa Cruz Biotechnology, California, USA, 1:1000), polyclonal anti-PDE3B antibody (Santa Cruz Biotechnology, California, USA, 1:3000), and monoclonal anti-GAPDH antibody (Sigma-Aldrich, St. Louis, MO, 1:10,000) at 4 °C overnight. After washing, the membrane was incubated with peroxidase-conjugated IgG (ZSJQ-BIO, Beijing, China, 1:10,000) for 1 h at room temperature. Final detection was performed using enhanced chemiluminescence detection solution 1 and 2 (1:1) (ECL, Millipore, Billerica, MA). Densitometry was used to measure the expression of PDE3A and PDE3B relative to GAPDH or α-actin with ImageJ (NIH).

Expression levels of COX-2, α-SMA, Col1A1, and VEGF were determined by immunoblotting after blocking with 5% nonfat milk, with goat anti-COX-2 antibody (1:500, Cayman Chemical, Ann Arbor, Michigan), a monoclonal antibody against α-SMA (Clone 1A4, 1:1000), a rabbit polyclonal antibody against Col1A1 (ORIGENE, Rockville, MD), or an anti-VEGF polyclonal antibody (1:2000, Millipore Temecula, CA), and visualized with HRP (horseradish peroxidase)-conjugated secondary antibodies using the SuperSignal West Pico Chemiluminescent substrate kit (Thermo Scientific, Rockford, IL). Monoclonal anti-GAPDH antibody (1:50,000 dilution, Sigma-Aldrich) was used as loading control.
Total RNA was isolated from cells and tumor samples using the QIAshredder and RNeasy Mini kit (Qiagen, Valencia, CA). cDNA was prepared using the iScript cDNA synthesis kit (Bio-Rad). cDNA samples were diluted 1:10 and real-time PCR was performed using IQ SYBR Green supermix and gene specific primers in the iCycler real-time PCR detection system (Quanta Bioscience, Gaithersburg, MD). All primers were designed using Beacon designer software 7.8 (PREMIER Biosoft, Palo Alto, CA). The expression of target RNA relative to the housekeeping gene HPRT1 was calculated based on the threshold cycle (Ct) as R = 2^-Δ(ΔCt), where ΔC_t= C_t of target - C_t of HPRT1.

Total protein was extracted from MDA-MB-231 cells grown in a 100 mm dish or from frozen MDA-MB-231 tumor tissue by using 1x cracking buffer [100 mmol/L Tris (pH 6.7), 2% glycerol] containing a protease inhibitor (Sigma) at 1:200 dilution. Protein concentration was estimated using the Bradford Bio-Rad protein assay Kit (Bio-Rad). Approximately 100 µg of total protein was used in each experiment. Expression levels of PD-L1 were determined by immunoblotting using a rabbit polyclonal against human PD-L1 at 1:1,000 dilution (GeneTex, Irvine, CA). Monoclonal anti-GAPDH antibody (1:50,000 dilution, Sigma-Aldrich) was used as loading control. Proteins were visualized with HRP (horseradish peroxidase)-conjugated secondary antibodies using the SuperSignal West Pico Chemiluminescent substrate kit (Thermo Scientific).

Pelleted erythrocytes were first frozen and then thawed in 20-fold volume of 5 mM phosphate buffer containing 150 mM NaCl, protease inhibitors (Roche) and phosphatase inhibitors (Sigma). Then, 200 µl were isolated and used as total lysate. The rest were centrifuged with 20,000 g for 20 min. Supernatant were removed and pellets were washed for four times before dissolving in the same buffer with 1% Triton X-100. 50 µg of membrane protein and 150 µg total protein were loaded for western blot detection of membrane bound and total GAPDH using monoclonal anti-GAPDH antibody (Sigma-aldrich) (1:1000 in blocking buffer).

Mouse erythrocytes were fixed with 100% ice-cold methanol for 10 min. The fixed cells were washed two times with PBS, blocked by 1% BSA in PBS (Blocking buffer, pH 7.4.) for 1 h at room temperature. Cells were incubated with monoclonal anti-GAPDH antibody (Sigma-Aldrich, 1:100 in blocking buffer) at 4 °C for overnight. The cells were washed three times with PBS, incubated with Alexa fluor 594 donkey anti-mouse IgG(H+L) (1:1,000 dilution) or Alexa fluor 488 donkey anti-rabbit IgG(H+L) (1:1,000 dilution) (Life Technologies) for 1 h at room temperature in dark, then washed three times and re-suspended in PBS. Cell-smear were made and dried in dark. The slides were mounted with cover glass by mounting medium (VECTASHIELD H-1400, Vector, CA). Pictures were taken under Zeiss LSM 780 confocal microscope (Carl Zeiss Inc., Jena, Germany).