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4 protocols using itga6

1

Western Blot Analysis of Cell Lines

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25 μg lysate from control or rudhira knockdown cell lines of SVEC was used for western blot analysis by standard protocols. Blots were cut into strips and incubated with primary antibodies as indicated: MMP2, MMP9 (Cell Signaling Technology), Fibronectin, Vimentin, GAPDH (Sigma Chemical Co., USA), Sox 9 (RND Systems), Laminin, ColIV, Wnt3a (Abcam), Itga6, Itga4 (BD Biosciences) and BCAS3 (Bethyl Labs, USA). HRP conjugated secondary antibodies against appropriate species were used and signal developed by using Clarity Western ECL substrate (Biorad, USA).
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2

Multicolor Immunofluorescence Staining Protocol

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Immunofluorescence was performed as described previously2 (link). The following antibodies or chemical compounds were used: COL17A1 (Abcam, ab186415, 1:400), Keratin 14 (Biolegend, 906004, 1:500), ITGA6 (BD, 555734, 1:200), Keratin 15 (Biolegend, 833904, 1:300), NFκB (Santa Cruz, sc-101749, 1:50), CD11b (BD, 557686, 1:100), F4/80 (Bio-Rad, MCA497A488, 1:100), KI67 (Invitrogen, 14-5698-82, 1:300), CD3 (Biolegend, 100235, 1:100), MHC2 (Biolegend, 107605, 1:100), CD31(BD Biosciences, 557355, 1:200), anti-DNA/RNA Damage (Abcam, ab62623, 1:2000), Tom20 (Thermo Fisher, 11802-1-AP, 1:30), Keratin 1 (Abcam, ab185628, 1:300), survivin (Cell signaling, 2808, 1:100), casp3 (Cell signaling, 9661, 1:200), Tuj1 (Covance, PRB-435P, 1:500), CD34-FITC (Invitrogen, 11-0341-85, 1:100), ITGA6-PE (BD, 555736, 1:100), Sca1-APC (Miltenyi Biotec, 130-102-343, 1:100), CD45-APC-cy7 (BioLegend, 103116, 1:100). FV10-ASW4.2 was used for capturing confocal images. CellSens Standard v1.13 (Olympus) was used for collecting HE data.
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3

Comprehensive Antibody Panel for Cell Signaling

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Those obtained from Cell Signaling Technology included CCND1 (E3P5S XP), HRAS (D2H12), VIM (D21H3 XP), CLDN1 (D5H1D XP), β-CATENIN (D10A8 XP), SNAIL (C15D3), CDH1 (24E10), ZEB1 (E2G6Y XP), RELA (p65) (D14E12 XP), STING (D1V5L), TBK1 (D1B4), IRF3 (D83B9), RELB (C1E4), IKBA (44D4), NF-KB2 (p100/p52) (4882), H3 (D1H2 XP). Other antibodies included HEY1 (ProteinTech 19929–1-AP), ITGA6 (BD Horizon GoH3, BV650), ITGB1 (BioLegend HMB1–1 APC/Cy7).
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4

Profiling PGCLCs and SSCLCs by Flow Cytometry

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PGCLCs were dissociated and stained with ITGA6 (BD, USA) and EpCAM (BD, USA) primary antibodies (Additional file 4: Table S2) [25 (link)]. SSCLCs were dissociated with Accutase (Thermo Fisher Scientific, USA) and stained with PLZF (Invitrogen, USA) primary antibody (Additional file 4: Table S2) after fixing, permeabilizing, and blocking processes [26 (link)]. The percentages of PGCLCs and SSCLCs were determined by a flow cytometer (Beckman Coulter, USA).
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