Irdye 800cw goat anti rabbit igg h l

Cells were lysed using tissue protein extraction reagent (Thermo, USA) containing phosphatase inhibitor cocktail (1:100, Thermo), protease inhibitor cocktail (1:100, Thermo), and 5 mM EDTA (Thermo); lysates were oscillated and centrifuged (13,000×g, 15 min, 4 °C). The supernatant was collected and stored at − 80 °C. The protein concentrations were determined using the BCA protein assay kit (Thermo). The protein was mixed with 5X SDS-PAGE protein loading buffer (Beyotime, China) and denatured by 100 °C water bath. Then, the samples denatured were electrophoresed in 10% SDS-PAGE and transferred to PVDF membranes using transfer device (Bio-Rad). The membranes were blocked with 5% non-fat milk prepared in TBST for 1 h at 37 °C and then incubated at 4 °C overnight with the primary antibodies: ERK1/2 (1:1000, Cell Signaling Technology, p44/42 MAPK Rabbit mAb, 4695), p-ERK1/2 (1:1000, Cell Signaling Technology, Phospho-p44/42 MAPK (ERK1/2) XP®, Rabbit mAb, 4370), and the internal normalization mouse anti-GAPDH (1:1000, Santa Cruz Biotechnology). Next, the membranes were washed in TBST, incubated with secondary antibody: Goat anti-Rabbit IgG (H + L) IRDye 800CW or Goat anti-Rabbit IgG (H + L) IRDye 800CW (1:20,000; LI-COR) for 1 h at 37 °C. The images were observed with a UVA Bio Imaging System and analyzed with ImageJ software.

The procedure of Western blot analysis was performed as previously described [5 (link)]. Total protein content was isolated from HASM cells using a protein extraction lysis buffer (Biyuntian, China) in the presence of a protease inhibitor (PMSF, Biyuntian, China). The protein concentration of the cell lysates was quantified using a BCA protein assay kit (Biyuntian, China). An equal amount of protein derived from the different groups was separated on a 10% SDS gel. Following separation, the proteins were transferred to a nitrocellulose membrane (PALL, USA) and the membranes were incubated at room temperature in 5% non-fat milk in TBST. Subsequently, the membranes were incubated with rabbit polyclonal Abs against GAPDH and a-SMA (Immunoway, USA) and rabbit monoclonal Abs against AKT and phosphorylated AKT (Cell Signaling Biotechnology). The following morning, the membranes were incubated with a secondary antibody (goat anti-rabbit IgG H&L IRDye 800CW, Licor, USA) at room temperature for 1 h. The membranes were quantified by densitometry using the Odyssey infrared image system (Licor, USA). GAPDH was used as the control for the normalization of the relative protein expression levels.

Anti-HA-tag (rabbit polyclonal, MilliporeSigma, Burlington, MA, USA), anti-FLAG-tag (mouse monoclonal, MilliporeSigma, Burlington, MA, USA), anti-HA-tag (mouse monoclonal, MilliporeSigma, Burlington, MA, USA), anti-HBc (rabbit polyclonal, DAKO, Carpinteria, CA, USA), and anti-HBc (rabbit monoclonal, Gilead Sciences, Inc., Foster City, CA, USA) were used as primary antibodies. As secondary antibodies conjugated with horseradish peroxidase (HRP), we used goat anti-mouse (MilliporeSigma, Burlington, MA, USA) and goat anti-rabbit (MilliporeSigma, Burlington, MA, USA). For visualization, we used a SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific, Waltham, MA, USA) and LAS-4000 imager. As secondary antibodies for the LI-COR system, we used goat anti-rabbit IgG (H + L) IRDye 800CW (LI-COR Biosciences, Lincoln, NE, USA) and goat anti-mouse IgG (H + L) IRDye 680RD (LI-COR Biosciences, Lincoln, NE, USA). Western blots were visualized using an LI-COR Odyssey CLx system and the Image Studio Lite Software (LI-COR Biosciences, Lincoln, NE, USA).