Species specific secondary antibodies

Cells lysis was performed with total protein extraction buffer (NCM Biotech, Suzhou, China), and tissues were homogenized in T-PER tissue protein extraction reagent (Thermo Fisher Scientific), to which a combination of protease and phosphatase inhibitors was added (NCM Biotech, Suzhou, China). The protein concentration was measured by a BCA Protein Assay Kit (Thermo Fisher Scientific). Cell or tissue lysates (approximately 20 μg of protein) were subjected to SDS-PAGE (8–10%) and transferred to nitrocellulose (NC) membranes. After blocking with 5% fat-free milk, the NC membranes were incubated with different primary antibodies at 4 °C overnight. Antibodies against the following proteins were used: GPAA1 (1:1000, Bioss, bs-13496R), caveolin-1 (1:1000, Abcam, ab2910), AP2B1 (1:1000, Proteintech, 15,690–1-AP), β-actin (1:10000, Abcam, ab5644), EGFR (1:1000, Abcam, ab52894), p-EGFR (Y1068) (1:1000, Abcam, ab40815); ERBB2 (1:1000, Abcam, ab16901); p-ERBB2 (Y877) (1:1000, Abcam, ab47262), p-AKT (S473) (1:1000, CST, #4060), and AKT (1:1000, CST, #4685). After three washes in TBST (pH 7.4), membranes were incubated with species-specific secondary antibodies (Thermo Fisher Scientific) at room temperature for 1 h. Ultimately, protein bands were visualized by an Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, USA).

Proteins were extracted using RIPA buffer (.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40 and 10 mM EDTA, Millipore) and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Anti-ERO1L (ab177156, Abcam), β-actin (ab8226, Abcam,) Vimentin (Servicebio, GB111308), and E-cadherin (Servicebio, GB13083) antibodies were used. β-Actin was used as the loading control. The next day, the membranes were incubated with species-specific secondary antibodies (ThermoFisher Scientific, USA). Finally, bound secondary antibodies were detected by Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, USA).

Whole-cell extracts were extracted using a total protein extraction buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors and the protein concentration was measured using a BCA Protein Assay Kit (Pierce Biotechnology, USA). Protein lysates were separated by 6-12% SDS-PAGE and transferred to a nitrocellulose membrane (Millipore, MA, USA). After blocking with 5% skimmed milk, the membrane was probed with one of the following primary antibodies: ERO1L (1:1000, Abcam, ab177156), HIF1α (1: 1000, Abcam, ab113642), BiP (1: 1000, Cell Signaling Technology, #3177), CHOP (1: 1000, Cell Signaling Technology, #2895), phospho-eIF2α (Ser51) (1:1000, Cell Signaling Technology, #3398), eIF2α (1:1000, Cell Signaling Technology, #5324), and β-actin (1:2000, Abcam, ab8226). The next day, the membranes were incubated with species-specific secondary antibodies (ThermoFisher Scientific, USA). Finally, bound secondary antibodies were detected by Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, USA).

Whole-mount in situ hybridization (WISH) was performed as previously described (Pearson et al., 2009; (link)
Sasidharan et al., 2013; (link)
King and Newmark, 2013 (link)). miRNA-based WISH was carried out using digoxigenin-labeled miRCURY LNA probes obtained from Exiqon. For immunostaining, animals were fixed using Carnoy's solution as described (Sánchez Alvarado and Newmark, 1999 (link)). Rabbit anti-ARRESTIN (1:5000, clone VC-1; gift of Dr Kiyokazu Agata, Kyoto University, Japan) and mouse anti-SYNORF-1 (1:100, DHSB) primary antibodies were used. Species-specific secondary antibodies were obtained from Molecular Probes (1:400). Hoechst 33342 (25 µg/ml; Sigma) was used as a nuclear counterstain. Animals were mounted with Mowiol mounting medium (Sigma) and stored at 4°C until imaging.

Cultured cells were fixed in 4 % paraformaldehyde followed by 0.25 % triton incubation. Samples were then rehydrated in phosphate-buffered saline, blocked with 1 % bovine serum albumin/10 % normal goat serum, and incubated with primary antibodies for 1 hour at room temperature that included: rat anti-mouse podocalyxin (1 μg/ml; R&D Systems), mouse anti-E-cadherin (0.25 μg/ml; BD Biosciences), rabbit anti-ZO-1 (2.5 μg/ml; Invitrogen-Zymed Laboratories), mouse anti-cytokeratins (clones AE1/AE3, 1:1000; Dako), rabbit anti-ezrin (1 μg/ml; Cell Signaling Technology Inc.), and mouse anti-Muc1 (1:500; gift from Dr John Stingl, Cambridge Research Institute, Cambridge, UK)). Cells were then incubated with fluorescently conjugated species-specific secondary antibodies (Invitrogen-Molecular Probes, Burlington, ON, Canada), and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (Sigma), mounted in glycerol containing the anti-fade agent diazabicyclo[2.2.2]octane (Sigma), and imaged using an Olympus FV1000 confocal microscope (Olympus) followed by processing using FV1000 Fluoview (Olympus) and Adobe Photoshop v12.0 software (Adobe, San Jose, CA, USA).