Goat pab to rb igg hrp

After ALV-J infected DF-1 cells for 48 h, the cells were collected, and then the cells were lysed with RIPA and the supernatant was collected. The protein concentration was then measured using a BCA protein concentration assay kit (Beyotime, Shanghai, China), and finally, a western blot analysis was performed.
A 12% sodium dodecyl sulfate-polyacrylamide (SDS) gel (GenScript, Nanjing, China) was used for protein separation. Then, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Polyvinylidene Fluoride) following 1-h blocking with 5% defatted milk. Then, primary antibodies were added and incubated at 4 °C overnight. After washing with tris-buffered saline tween (TBST), secondary antibodies were added for 1-h incubation at RT. Primary antibodies, including Mouse anti-gp85, JE9, donated by Professor Aijian Qin of Yangzhou University, and Rabbit anti-β-Actin were purchased from Novus Biologicals (Englewood, CO, USA). Secondary antibodies, including HRP Goat Anti-Mouse IgG(H+L), purchased from ABclonal (Wuhan, China), and Goat pAb to Rb IgG (HRP), purchased from Abcam (Cambridge, UK).

Twenty-four hours after irradiation, cell lysates were prepared using RIPA buffer (Santa Cruz Biotechnology, Dallas, TX, USA). Proteins were fractionated using 10–15% SDS polyacrylamide gradient gels, and the proteins were electrophoretically transferred to PVDF membranes (Millipore, Burlington, MA, USA). Primary antibodies were against S100A4 (Sheep IgG, Cat. # AF4138, R&D Systems, Minneapolis, MN, USA), p-Chk1 (Ser345) (Rabbit IgG, Cat. # 2348, Cell Signaling, Danvers, MA, USA), Chk-1 (Mouse IgG, Cat. # 2360, Cell Signaling) (dilute to 1:1000), and β-actin (Mouse IgG, Cat. # ab8226, Abcam, Cambridge, UK) (dilute to 1:10000). Secondary antibodies were used Goat pAb to Ms IgG (HRP) (Cat. # ab97023, Abcam) and Goat pAb to Rb IgG (HRP) (Cat. # ab97051, Abcam) at a 1:2000 dilution. PD407824 inhibited the phosphorylation and activity of Chk1-Ser345 [12 ], and siChk1 downregulated the expression of Chk1 and p-Chk1 [13 (link)]. Membranes were washed in PBST, and luminescence detected using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) was measured using an ImageQuant LAS 4000 (GE Healthcare, Little Chalfont, UK).