Horseradish peroxidase hrp conjugated secondary antibody

Western blotting was performed using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system as described previously [12 (link)]. Immunoblotting was performed by incubation at 4 °C with antibodies against CUL4A (1:1000; CST) and β-actin (1:2000, Santa Cruz Biotechnology) overnight. Blots were then washed and incubated with a 1:2000 dilution of horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson, West Grove, PA, USA), followed by three washes with Tris-buffered saline-containing Tween 20. Pierce Enhanced chemiluminescent HRP substrate (Thermo Fisher) was used for detection according to the manufacturer’s instructions.

SDS-PAGE was performed using a 15 % running gel on the Power-Pac Basic (Bio-Rad, USA). Briefly speaking, the protein samples were loaded on the gel and were electrophoresed at 120 V for 1.5 h. The bands of proteins on the gel were visualized by staining with Coomassie brilliant blue R-250. In Western blotting experiments, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes at 200 mA for 60 min. The membranes were first blocked in PBS containing 10 % (w/v) non-fat milk, and then incubated with a polyclonal antibody of mouse Retn (R&D, USA), diluted at 1:2000 in PBS containing 0.05 % Tween 20 (PBST) and 5 % (w/v) non-fat milk. After a washing, the membrane was incubated in the presence of horseradish peroxidase (HRP) conjugated secondary antibody (Jackson immuno, USA) diluted at 1:2 000 in PBST with 5 % (w/v) non-fat milk powder. All the incubating procedures were performed at 37 °C for 1 h or 4 °C for overnight with gentle shaking and the membranes were washed for three times before each incubation. Finally, the membrane was thoroughly rinsed five times with PBST for 5 min each, and then the rmResitin protein was visualized through enhanced electrogenerated chemiluminescence (ECL) reaction on X-ray film using western blotting substrate reagent (Thermo, USA).

Cells harvested on the 4, 9, and 14 days of differentiation were lysed on ice for 20 min with a radioimmunoprecipitation assay (RIPA) lysis buffer containing a protease inhibitor cocktail (GenDEPOT, Harris County, TX, USA). The lysates were centrifuged at 12,000 rpm for 30 min at 4 °C and transferred to a new 1.5 mL tube. The protein concentrations were measured using a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were incubated with 5% skim milk at room temperature for 1 h, then incubated overnight at 4 °C with primary antibodies, anti-alpha-SMA (Abcam, Cambridge, UK), anti-CNN1 (Abcam), and anti-SM-MHC (Abcam). After washing extensively, the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 2 h. The signal was detected using WESTSAVE (AbFrontier, Korea) and an enhanced chemiluminescence system. ImageJ (https://imagej.nih.gov/ij/,download.html, accessed on 14 September 2021) software was used to quantify the band intensity of the Western blot.

Antibodies were purchased from BD Transduction Laboratories (Heidelberg, Germany; E-cadherin clone 36; IF 1:100, IB 1:1000), Sigma-Aldrich (Munich, Germany; AJAP1 HPA012157, IF 1:100, WB 1:1000; SMA clone 1A4, IF 1:100; pan-cadherin CH-19, IB 1:1000; myc C3956, IF 1:100, IB 1:1000), Ambion (Darmstadt, Germany; GAPDH clone 6C5, IB 1:10,000), Thermo scientific (Darmstadt, Germany; Grp94 clone 9G10.F8.2, IB 1:500), Abcam (Cambridge, UK; AJAP1 ab121361, IF 1:100, IB 1:1000), R&D Systems (Wiesbaden, Germany; AJAP1 AF7970), AntibodyVerify (Las Vegas, NV, USA; AJAP1 AAS47449C), Santa Cruz (Heidelberg, Germany; β-catenin H102, IB 1:1000; β-casein M-14, IB 1:1000) and custom made antibodies against shrew-1 (Nanotools, IB 1:500, polyclonal Genovac and monoclonal Genovac clone F, IF 1:50 and IB 1:500). Secondary antibodies (Alexa Fluor 488-, 594- and 647-labeled antibodies, IF 1:400) were purchased from Molecular Probes (Leiden, The Netherlands) or Abcam (Cambridge, UK) and Horseradish peroxidase (HRP)-conjugated secondary antibody from Jackson Immuno Research (Dianova, Hamburg, Germany) or Biosource (Camarillo, CA, USA).