Anti goat igg hrp

Cell lysates were prepared by incubation with a lysis buffer (40 mM Tris-HCl pH 8.0, 120 mM NaCl, 0.1% Nonidet-P40) supplemented with protease inhibitors. Then proteins were then subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) before being transferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL). Primary antibodies were used to detect the relevant protein, and β-actin was used as loading control. Blots were developed with horseradish peroxidase (HRP)-conjugated secondary antibodies and proteins were visualized using enhanced chemiluminescence (ECL) (Amersham, Arlington Heights, IL), according to the manufacturer's protocol. Secondary antibodies, anti-mouse IgG-HRP, anti-goat IgG-HRP and anti-rabbit IgG-HRP were purchased from Santa Cruz biotechnology (CA, USA).

Cells were washed in ice‐cold PBS and sedimented at 1000 g for 10 min at RT. Pellets were suspended in RIPA buffer (10 mm Tris/HCl, pH 7.4, 1% sodium deoxycholate, 1% Triton X‐100, 0.1% SDS) containing 1 mm PMSF and protease inhibitor cocktail (Sigma‐Aldrich). Protein concentration was quantified using Bicinchoninic acid (Thermo Scientific, Rockford, IL, USA). Total proteins were separated by SDS/PAGE and transferred to Porablot NCP membranes (Macherey‐Nagel, Düren, Germany). Blots were probed with anti‐FLAG (1 : 2000; Sigma‐Aldrich), anti‐E‐cadherin (1 : 2000; Cell Signaling Technology, Danvers, MA, USA), anti‐N‐cadherin (1 : 2000; Cell Signaling Technology), anti‐Snail (1 : 2000; Cell Signaling Technology), and β‐actin (1 : 2000; Santa Cruz Biotechnology, Dallas, TX, USA) antibodies. Primary antibody binding was detected with anti‐goat IgG‐HRP (1 : 2000; Santa Cruz Biotechnology), anti‐mouse IgG‐HRP (1 : 2000; Santa Cruz Biotechnology), or anti‐rabbit IgG‐HRP (1 : 2000; Santa Cruz Biotechnology). Membranes were revealed using the EZ‐ECL chemiluminescence kit (Biological Industries, Haemek, Israel) and the ChemiDoc Touch Gel Imaging System (Bio‐Rad, Hércules, CA, USA).

Cell lysates were extracted from the cell pellet using lysis buffer (40 mM Tris-HCl pH 8.0, 120 mM NaCl, 0.1% Nonidet-P40) supplemented with protease inhibitors. Proteins in whole-cell lysates were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Amersham, Amersham, UK). nitrocellulose membranes were blocked with 5% skim milk in phosphate-buffered saline containing Tween 20 and incubated with primary antibodies overnight at 4°C. The blots were then incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies and proteins were visualized by enhanced chemiluminescence (Amersham), according to the manufacturer’s protocol. Secondary antibodies, anti-mouse IgG-HRP, anti-goat IgG-HRP, and anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology.

Antibodies to p-ERK1/2, ERK1/2, p-T308-AKT, AKT, p-Y705-STAT3, JAK1 were purchased from Cell Signaling Technology (Beverly, MA, USA). STAT3 and p-T1022-JAK1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies to HAS2 and C5R1 were purchased from Abcam (Cambridge, UK). 4,6-diamidino-2-phenylindole (DAPI) were purchased form Sigma (St Louis, MO, USA). rh-C5a protein and antibodies to CD105 and C5a were purchased from R&D Systems (Minneapolis, MN, USA). Chemical inhibitors specific to MEK (U0126), PI3K (LY294002), JAK1 (P6) and STAT3 (WP1066) were purchased from Calbiochem (San Diego, CA, USA). Anti-Goat Alexa Fluor 488, anti-mouse Alexa Fluor 546 were purchased from Invitrogen (Carlsbad, CA, USA). Anti-mouse IgG-HRP, anti-goat IgG-HRP and anti-rabbit Ig-HRP were purchased from Santa Cruz biotechnology (Santa Cruz, CA, USA). HA (Low molecular weight; 15-40 kDa) was purchased from R&D systems (Minneapolis, MN, USA).

The extracts of primary cultures taken at 36 h or 8 days after plating were subjected to standard procedures. The protein amount was determined using BioRad protein assay. Equal amounts of protein (30 µg) were electrophoresed on 10% acrylamide gel and electrotransferred to polyvinylidene fluoride membrane, followed by immunoblotting with antibodies for myosin heavy chain (MyHC; 1:1000, GTX73432, GeneTex, Taiwan), phospho-TAK1 (1:1000, Cell signaling, #4508), p-p38 (1:1000, Cell signaling, #4511), p-JNK (1:1000, GTX52328, GeneTex, Taiwan), p-ERK (1:1000, Cell signaling, #4370), and p-NF-kB (1:1000, Cell signaling, #3033). Goat anti-actin-HRP (1:5000, Santa Cruz Biotechnology) was used for protein quantification. The membrane was washed and incubated with secondary anti-mouse IgG-HRP (1:3000, sc-2005, Santa Cruz, California, USA), anti-goat IgG-HRP (1:3000, sc-2020, Santa Cruz, California, USA), and anti-rabbit IgG-HRP (1:3000, GTX213110-01, GeneTex, Taiwan). Detection was performed using the T-Pro LumiLong Plus Chemiluminescence Detection Kit (JT96-K004M, T-Pro Biotechnology, Taiwan) and the blots were directly processed on an Amersham Imager 600 (GE, Boston, USA). Densitometry was performed using ImageJ.

Blots were probed with the primary antibodies anti-GFP (1:2000) and anti-GST (1:2000) from Sigma; anti-actin (1:2000) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-ECE-1 (1:1000) from Abcam (Cambridge, UK). Detection was performed using secondary anti-goat IgG-HRP (1:2000) or anti-rabbit IgG-HRP (1:2000) from Santa Cruz Biotechnology, and the EZ-ECL chemiluminiscence kit from Biological Industries (Kibbutz Beit, Haemek, Israel).