Anti gdnf antibody

1 ml ASC-CM was incubated overnight at 4°C with 2 μl anti-GDNF antibody (500μg/ml) (Abcam, Cambridge, MA). Protein A-agarose was added, and a second overnight incubation was performed, followed by centrifuging.

Cells were treated with RIPA lysis buffer for 15 min prior to total protein extraction. Protein concentration was determined by BCA assay. Samples (20 μg) were subject to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, USA). The membrane was blocked with 5% bovine serum albumin (BSA) for 1 h and then incubated with the anti-GDNF antibody (1: 1000; Abcam, Cambridge, USA) or anti-GAPDH antibody (1:1000; Abcam), followed by incubation with the corresponding HRP-conjugated secondary antibody (1:500 ; Beyotime). The protein bands were visualized using an ECL assay kit (Beyotime) and quantified using ImageJ (National Institutes of Health, Bethesda, USA). GAPDH was used as the loading control.

Total proteins were extracted and quantified. Protein was separated by 10% SDS‐PAGE and transferred onto a PVDF membrane. The membrane was incubated with primary antibody at 4°C overnight, then incubated with the secondary antibody for 1 hour at room temperature. The immunoblots were detected by an enhanced chemiluminescent substrate (Millipore). Antibodies: anti‐RET antibody (1:1000; Abcam), anti‐Lgr5 antibody (1:1000; Abcam), anti‐β‐catenin antibody (1:1000; Proteintech, USA), anti‐TPH1 antibody (1:500; Abcam), anti‐GDNF antibody (1:500; Abcam), anti‐Ngn3 antibody (1:300; Abcam), anti‐β‐actin antibody (1:1000; Zhongshan Gold Bridge, Beijing, China), goat anti‐mouse IgG (1:1000; Zhongshan Gold Bridge) and goat anti‐rabbit IgG (1:1000; Zhongshan Gold Bridge).