Hrp conjugated goat anti rabbit igg antibody sc 2004

Antibodies against VEGFR1 (#ab32152), VEGFR2(#ab24313), NRP1(#ab81321), E-cadherin(#ab40772), N-cadherin(#ab76057), Snail(#ab229701), fibronectin(#ab32419), TWIST1(#ab50887), GAPDH(#ab181602), CD34(#ab81289), vimentin(#ab92547) and ZEB1(#ab203829) were purchased from Abcam (Cambridge, UK). An HRP-conjugated goat anti-rabbit IgG antibody (#sc-2004) was purchased from Santa Cruz Biotechnology. According to the manufacturer’s instructions, the total protein of GC tissues and transfected cells was extracted with RIPA lysis buffer (Beyotime, China). After the protein concentration was determined by the BCA protein assay, the equivalent amounts of cell lysates were electrophoresis with 10% SDS polyacrylamide gel and transferred onto polyvinylidene membrane, which was then placed on 5% skim milk prepared with TBS-T and sealed at 4 °C for 1 h. After that, the corresponding primary antibodies were used to incubate the blots. Incubation of blots with HRP-labelled secondary antibodies prior to visualise with enhanced chemiluminescence reagents (Millipore, Billerica, MA, USA).

U937 cells were co-cultured with bacteria under standard conditions following specified time point, then cells were washed with 1X PBS pH 7.4, and total proteins were extracted using RIPA buffer (Thermo Fisher Scientific, MA, USA). The expression levels of LC3-II and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) were determined by western blotting using a rabbit anti-LC3 polyclonal antibody (2775S; Cell Signaling Technology, MA, USA) and a mouse anti-GAPDH monoclonal antibody (611,463; BD Pharmacia, San Jose, CA, USA), and followed by a Horseradish peroxidase (HRP)-conjugated goat anti- rabbit IgG antibody (sc2004; Santa Cruz Biotechnology, Inc., TX, USA) and a HRP-conjugated goat anti-mouse IgG antibody (sc2005; Santa Cruz Biotechnology, Inc., TX, USA). The signal was visualized using Pierce^® ECL Western blotting substrate (Thermo Fisher Scientific, MA, USA).