Western bright chemiluminescence reagent

For western blot analyses, PBS‐washed cells were lysed in RIPA buffer plus supplements [52 (link)] for 60 min at 4 °C on a shaker and afterward centrifuged at 10 000 g at 4 °C for 30 min. Protein concentration was measured by BCA assay (Thermo Scientific) according to the manufacturer's protocol. Equal amounts of protein extracts were separated on a 10–12% SDS/PAGE and transferred onto nitrocellulose membranes. Membranes were incubated with primary antibodies against L1 ectodomain (1 : 11 000; L4343‐25ul; Sigma‐Aldrich), L1 (1 : 1000; ab24345; Abcam, Cambridge, MA, USA), ADAM10 (1 : 1000; #14194; Cell Signaling, Danvers, Ma, USA), ADAM17 (1 : 1000; ab6326; Abcam, Cambridge, UK), Ezrin (1 : 1000; sc‐58758; Santa‐Cruz, Dallas, TX, USA), galectin‐3 (1 : 1000; #12733; Abcam), FGFb (1 : 1000; EPR20145‐227; Abcam), and β‐actin (1 : 1000; #4967; Cell Signaling) at 4 °C overnight. Species‐specific HRP‐conjugated secondary antibodies (goat anti‐rabbit; P0448; DAKO and rabbit anti‐mouse; P0260; DAKO, Glostrup, Denmark) were used in dilutions of 1 : 10 000 at room temperature for 1 h. The HRP signal was detected by adding Western Bright Chemiluminescence Reagent (Advansta, San Jose, CA, USA).

Equal volumes of AH samples and cell culture supernatants were separated on a 12% SDS-PAGE and transferred onto nitrocellulose membranes. The primary antibodies used (incubated overnight at 4 °C) were TFF1 (1:1000 abcam, Cambridge, UK, # ab92377) and ß-actin (1:1000): #4967, Cell Signalling Technology (Cambridge, UK). The secondary antibody used was HRP-conjugated goat-anti-rabbit antibody (1:10,000); P0448; Dako (Glostrup, Denmark). Signals were developed by the WesternBright Chemiluminescence Reagent (Advansta, San Jose, CA, USA).

After washing in PBS, cells were lysed for 60 min at 4 °C in RIPA buffer plus additives (see: [15 (link)]) and cleared by a 10 min centrifugation step at 14,000 rpm and 4 °C. Equal amounts of protein extracts were separated on a 12% SDS-PAGE and transferred onto nitrocellulose membranes. Primary antibodies used (incubated overnight at 4 °C): EMP1 (1:1000): ab230445, Abcam; p53 (1:400): sc-71817, Santa Cruz; TFF3 (1:500): TA307376, Origene; ß-actin (1:1000): #4967, Cell Signalling. Secondary antibodies used: HRP-conjugated goat-anti-rabbit or rabbit-anti-mouse antibody (1:10,000): P0448 and P0260; DAKO. Signals were developed by the Western Bright Chemiluminescence Reagent (Advansta).