Anti pan cadherin

LPS (E. coli LPS serotype 0111:B4), sodium pentobarbital, Evans Blue dye, collagenase and trypsin were purchased from Sigma (St. Louis, MO, USA). rh-omentin protein was purchased from GeneTex (Irvine, CA, USA). The following primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): anti-Scr, anti-phospho-Scr (Try416), anti-Akt, anti-phospho-Akt (Ser473), anti-eNOS, anti-phospho-eNOS (Ser1177), anti-NF-κB Rel and anti-phospho-NF-κB Rel (Ser536). Anti-VE-cadherin, anti-pan-cadherin and anti-cleaved caspase-3 antibodies were purchased from Abcam (Cambridge, UK). Anti-GAPDH, anti-CD31, anti-caspase-3, anti-GSK-3β and anti-phospho-GSK-3β (Ser9) antibodies were purchased from Bioworld Technology (Nanjing, China). Ad-β-gal and full-length human omentin (Ad-omentin) were constructed by Genechem (Shanghai, China). Ad-β-gal was used as a control.

For immunofluorescence only, cells were fixed and permeabilized as described in the “Cell culture and fixation” Section. Samples were first blocked at room temperature for 30 min in blocking buffer consisted of 4% (wt/vol) UltraPure BSA (Thermo Fisher Scientific) in 2× SSC supplemented with 3% (vol/vol) RNasin Ribonuclease inhibitor (Promega), 6% (vol/vol) murine RNAase inhibitor and 1 mg/ml yeast tRNA. Samples were then incubated with primary antibodies (anti-pan Cadherin, Abcam) in blocking buffer at a concentration of 2 μg/ml for 1 h at room temperature, and washed three times with 2× SSC for 10 min each. Samples were then incubated with oligo-labeled secondary antibodies in blocking buffer at a concentration of 3.75 μg/ml for 1 h at room temperature, then washed with 2× SSC three times for 10 min each. Samples were fixed again with 4% (vol/vol) PFA in 2× SSC for 10 min and washed three times with 2× SSC.
To combine MERFISH with immunofluorescence, samples were first stained with encoding probes and washed as described in the “Encoding probe staining” section above. To stabilize these probes, samples were then briefly post-fixed with 4% (vol/vol) PFA in 2× SSC at room temperature for 10 min and washed three times with 2× SSC. Samples were then stained for immunofluorescence as described above.

SH-sy5y cells were treated with 1000IU/mL hIFN-α-2b and harvested at 24 h. Cytomembrane proteins were extracted with the Mem-PER eukaryotic membrane protein extraction kit (Thermo Fisher Scientific, USA) according to the manufacturer’s recommendations66 (link). Anti-pan-cadherin (Abcam, USA) and anti-tublin (Abcam, USA) antibodies were applied to test the levels and the purity of membranal proteins67 .

Cell lysates were prepared by sonication in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 5 mM MgCl₂, 1% Triton X-100 and a complete protease inhibitor mixture tablet (Roche Applied Science). Equal amounts of protein (30–50 μg) were subjected to 6 or 10% SDS-PAGE and blotted onto a PVDF membrane. The membranes were then incubated with anti-IP₃R3 (1:1000; Abcam) or anti-Orai2 (1:1000; Abcam) and anti-pancadherin (1:1000; Abcam) or anti-β-actin (1:10000; Sigma) for loading controls and signals were detected with ECL reagent.

To measure protein levels, Western blotting (WB) was performed according to standard protocols. For analysis, the following primary antibodies were used; anti-CHP1 (Thermo Fisher Scientific; PA5-29876), anti-NHE1 (SCBT; 4E9), anti-PLS3 [Eurogentec (Oprea et al., 2008 (link)); 1238], anti-ACTB (Sigma, A5316), anti-GAPDH (Sigma, G-9295), anti-HSP90 (Cell Signaling; 4877), anti-EGFR (SCBT; sc-03), and anti-pan-cadherin (Abcam; ab6528).