Fibronectin ep5

Anti-LC3B, anti-Bcl-xL, anti-Atg16L1, anti-Rac1, anti-Rho1, anti-Cdc42, anti-phospho-ERK, anti-EEA1 and anti-LAMP1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse monoclonal antibodies reactive with LRP1 (11H4) were a kind gift from Dr. Strickland, University of Maryland School of Medicine, Baltimore. Fibronectin (EP5) and ubiquitin (P4D1) were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA); anti-Cx43, anti-ERK, anti-Bcl-2 and anti-RPTPβ antibodies were from BD Biosciences (Tokyo, Japan); anti-multi ubiquitin monoclonal antibody (FK1) was from MBL (Nagoya, Japan); anti-GAPDH antibody was from GeneTex (Irvine, CA, USA) and anti-LC3 (clone 1703) antibody was from Cosmo Bio (Tokyo, Japan). Anti-RPTPα rabbit polyclonal antibodies for immunoblotting were provided by Dr. Jan Sap; anti-α-tubulin and anti-FLAG M2 antibodies, N-acetyl-l-cysteine (NAC) and ammonium chloride were from Sigma Aldrich (St. Louis, MO, USA); ProLong Gold Antifade Reagent with DAPI and DIDS were from Invitrogen (Eugene, OR, USA); an ERK inhibitor, U0126, was from Cayman Chemical (Ann Arbor, MI, USA); Rac1 inhibitor, NSC23766 was from Wako Pure Chemical Industries (Osaka, Japan); and GSH was from Merck (Darmstadt, Germany).

Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors for 20 min. Proteins (20 μg) extracted from cells were separated on SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was blocked for 1 h with blocking buffer (5% non-fat milk in PBS containing 0.1% Tween 20) and then incubated with primary antibodies dissolved in blocking buffer at 4 ℃ overnight. Membranes were washed with TBS-T buffer (PBS with 0.1% Tween 20) for 5 min three times and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at RT for 1 h. The membrane was developed using an ECL Kit (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) with X-ray film.
The primary antibodies used in this western blotting were as follows: E-cadherin (H-108, 1:1000, sc-7870; Santa Cruz Biotechnology, Inc.), N-cadherin (H-63, 1:1000, sc-7939; Santa Cruz Biotechnology, Inc.), fibronectin (EP5, 1:1000, sc-8422; Santa Cruz Biotechnology, Inc.), vimentin (V9, 1:1000, sc-6260; Santa Cruz Biotechnology, Inc.), slug (1:1000, GTX31749; GeneTex), snail (1:10000, GTX125918; GeneTex), and GAPDH (D4C6R, 1:1000, 97,166; Cell Signaling Technology).

Crucial ECM proteins collagen and fibronectin were measured in all the sections of DT of PD and compared to NT. The primary antibody used for collagen was mammalian collagen I (1:100, COL1A1G3, Santa Cruz Biotechnology), and for fibronectin was mammalian fibronectin (1:100, Fibronectin-EP5, Santa Cruz Biotechnology). The secondary antibody used was anti-mouse IgGκ (1:50, BP-HRP, Santa Cruz Biotechnology) coupled to Horseradish peroxidase. Protease-induced epitope retrieval was carried out in accordance with literature recommendations for ECM (Rickelt and Hynes, 2018 (link)). The droplets of primary antibodies for each protein were added and incubated overnight at 4°C in a humid chamber. The following day, secondary antibody drops were added to each sample and incubated for 2 h in a humid environment. Slides were washed with 1XTBST buffer, dehydrated with ethanol, cleared in xylene, and mounted using DPX to be viewed under a microscope. Note: According to the quantification, ID tissues lacked considerable quantities of collagen; hence they were not tested for IHC imaging.