Horseradish peroxidase conjugated goat anti mouse secondary antibody

Tissue specimens or cells were lysed in a radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, P.R. China). Total protein concentration was assessed using a Bicinchoninic Acid Assay Kit (Beyotime Institute of Biotechnology). Equal amounts of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Subsequent to blocking with 5% nonfat milk in TBS containing 0.1% Tween 20 (TBST), the membranes were incubated overnight at 4°C with mouse anti-human monoclonal Notch1 antibody (1:1,000 dilution; sc-373944; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse anti-human monoclonal β-actin antibody (1:1,000 dilution; sc-69879; Santa Cruz Biotechnology). After washing with TBST, the membranes were probed with a goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:5,000 dilution; sc-2005; Santa Cruz Biotechnology), and protein signals were visualized with an ECL detection kit (GE Healthcare Life Sciences, Chalfont, UK). β-Actin was used as a loading control.

The primary antibodies mouse anti-human AEG-1 antibody (sc-517220), mouse anti-human phosphatase and tensin homolog (PTEN) antibody (sc-7974), mouse anti-human AKT antibody (sc-56878), mouse anti-human p-AKT antibody (sc-271966), and mouse anti-human GAPDH antibody (sc-81545) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Protein was isolated from tissue samples or cells using ice-cold lysis buffer (Beyotime Biotechnology, Jiangsu, P.R. China). A BCA Protein Assay Kit (Pierce; Thermo Fisher Scientific, Inc.) was applied to examine the concentration of total protein. Equal amounts of protein were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the fractionated proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Boston, MA, USA). After blocking with TBS/0.1% Tween (TBST) containing 5% skimmed milk at room temperature for 2 h, the membranes were incubated with the primary antibodies at 4°C overnight. The membranes were washed three times with TBST and then probed with goat anti-mouse horseradish peroxidase-conjugated secondary antibody for 2 h (Santa Cruz Biotechnology) at the dilution ratio of 1:5,000. The protein signals were visualized using an ECL prime Western blot substrate (GE, Amersham, UK). Relative protein expression was presented as the density ratio versus GAPDH.

Total protein was extracted from cultured cells using RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific, Waltham, MA, USA) and then quantified using a BCATM Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were separated on 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Beyotime Institute of Biotechnology, Shanghai, China). Following blocking by incubation with 5% skimmed milk, membranes were incubated overnight at 4 °C with mouse antibodies against MAPK1 (1:1,000 dilution; sc-81459; Santa Cruz Biotechnology Inc., Dallas, TX, USA) or GAPDH (1:1,000 dilution; sc-32233; Santa Cruz Biotechnology Inc.), followed by further incubation with goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:5,000 dilution; sc-516132; Santa Cruz Biotechnology Inc.) for 2 h at room temperature. Protein signals were visualized using an Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Billerica, MA, USA). GAPDH served as an internal control.

Transfected A549 cells were collected and washed with PBS for three times. The total protein of cells was extracted with the protein lysis buffer (Cell Signaling Technology Inc., Danvers, MA, USA), and the concentration of protein was determined using a bicinchoninic acid assay. Protein samples (40 µg) were separated on a 10% SDS-PAGE gel for 30 min and transferred to polyvinylidene difluoride membranes. Then, the membranes were blocked in tris-buffered-saline-Tween-20 (TBST; containing 0.1% Tween-20) with 5% skimmed milk for 2 h at room temperature and incubated with primary antibodies against HMGA2 (1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-130024) and β-actin (1:2,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-130065) at 4°C overnight with agitation. Following washing, the membranes were incubated with goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:2,000; Santa Cruz Biotechnology, Inc.; sc-516102) at room temperature for 2 h. Signals were visualized using an enhanced chemiluminescence luminescent reagent (cat. no. sw2030; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and were developed using a gel imaging system. β-actin was used as the internal reference. Bands were analyzed with Image J software (version 1.51j8; National Institutes of Health, Bethesda, MD, USA).

Total proteins were isolated from tissues or cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with protease inhibitor PMSF (Beyotime Institute of Biotechnology). The total protein concentration was detected by a BCA assay kit (Beyotime Institute of Biotechnology). Equal amounts of proteins were fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). After blocking in 5% nonfat milk in Tris-buffered saline-Tween (TBST) for 1 h, the membranes were incubated at 4°C overnight with primary antibodies against the following proteins: MAPK1 (sc-81459; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and GAPDH (sc-47724; Santa Cruz Biotechnology, Inc.). The membranes were subsequently washed with TBST three times and probed with goat anti-mouse horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. An electrochemiluminescence substrate Western blot detection system (Pierce; Thermo Fisher Scientific, Inc.) was added to visualize the protein blots. GAPDH served as a loading control.