Journal: Journal of cell science

Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis.

doi: 10.1242/jcs.223453

Figure Lengend Snippet: Fig. 1. EMC5 and EMC6 are essential for EMC maturation. (A) Schematic representation of the primary structure of all EMC subunits (EMC1–EMC10). Domains, boundary residue numbers and predicted glycosylation sites are indicated. Pyrrolo-quinoline quinone (PQQ) and tetratricopeptide repeats (TPR) are shown. (B) siRNA-mediated depletion of EMC1–EMC10 and non-targeting control (NTC) for 72 h in U2OS Flp-In™T-Rex™cells. Whole-cell lysates (WCL) of individually depleted cells were separated by SDS-PAGE and resulting western blots probed for each subunit and tubulin (TUB) as indicated. The asterisk (*) denotes a nonspecific band. (C) U2OS Flp-In™T-Rex™cells modified by CRISPR/Cas9 to knockout EMC6 (Δ6) were reconstituted by inducing expression of an empty vector control (EV), EMC5 or EMC6 (DOX 1 ng/ml, 72 h). MG132 was added to cells where indicated (5 µg/ml, 8 h). Samples were prepared as in B. TUB, tubulin; Ub, ubiquitin.

Article Snippet: The following antibodies were used in this study: ACC1 (Cell Signaling, Danvers, MA, rabbit pAb, #4190), 1:1000; AlexaFluor R488 anti-mouse IgG (H+L) (Invitrogen, donkey pAb, #A21202), 1:4000 [immunoblotting (IB)], 1:400 [immunofluorescence (IF)]; AlexaFluor R488 anti-rabbit IgG (H +L) (Invitrogen, goat pAb, #A11008), 1:4000 (IB), 1:400 (IF); anti-mouse IgG-HRP (Santa Cruz Biotechnology, goat, #sc-2005), 1:10,000; anti-rabbit IgG-HRP (Santa Cruz Biotechnology, goat, #sc-2030), 1:10,000; EMC1 (rabbit pAb, kind gift of Enilza Espreafico, Sao Paulo, Brazil), 1:1000; EMC2 (Proteintech, Rosemont, IL, rabbit pAb, #25443-1-AP), 1:2000; EMC3 (Abcam, Cambridge, UK, rabbit pAb, #ab175537), 1:500; EMC4 (Abcam, rabbit pAb, #ab123719), 1:1000; EMC5/MMGT1 (Abcam, rabbit pAb, #ab174366), 1:1000; EMC6 (Abcam, rabbit pAb, #ab84902), 1:1000; EMC7 (Abgent, San Diego, CA, rabbit pAb, #AP11145c), 1:500; EMC8 (Abcam, rabbit pAb, #ab180065), 1:500; EMC9 (Abgent, rabbit pAb, #AP5632b), 1:500; EMC10 (Abcam, rabbit pAb, #ab180148), 1:1000; HA (purified from hybridoma, mouse mAb, clone 12CA5), 1:1000; histone H3 (Abcam, rabbit pAb, ab1791), 1:5000; HMGCR (Atlas Antibodies, Bromma, Sweden, mouse mAb, clone CL0260), 1:1000; Hsp70 (Santa Cruz Biotechnology, goat pAb, clone K-20, #sc-1060), 1:1000; LDLR (R&D Systems, Minneapolis, MN, goat pAb, #AF2148), 1:1000; Rap1a (Santa Cruz Biotechnology, rabbit pAb, clone C-17, #sc-1482), 1:1000; SCD1 (Abcam, mouse mAb, clone CD.E10, #ab19862), 1:1000; SM (Proteintech, rabbit pAb, #12544-1-AP), 1:1000; SOAT1 (Santa Cruz Biotechnology, mouse mAb, clone A-11, #sc-136959), 1:1000; SQS (Abcam, rabbit mAb, #ab109723), 1:2000; SQS (Abcam, rabbit mAb, #ab195046), 1:200 (IF), 1:2000 (WB); SREBP2 (R&D Systems, goat pAb, #AF7119), 1:1000; tubulin (Sigma-Aldrich, mouse mAb, #T5168/b-5-1-2), 1:1000; and ubiquitin (Cell Signaling Technologies, rabbit pAb, #3933S), 1:1000.

Techniques: Residue, Glycoproteomics, Control, SDS Page, Western Blot, Modification, CRISPR, Knock-Out, Expressing, Plasmid Preparation, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Defective EMC1 drives abnormal retinal angiogenesis via Wnt/β-catenin signaling and may be associated with the pathogenesis of familial exudative vitreoretinopathy

doi: 10.1016/j.gendis.2022.10.003

Figure Lengend Snippet: Loss of Emc1 in mouse endothelial cells results in retinal vascular defects. (A) Representative overview (top panels) and high-magnification images (bottom panels) of P7 control (Ctrl) and Emc1 iECKO/iECKO mouse retinae stained with Isolectin B4 (IB4). The circles indicate vessel outgrowth of Ctrl retinae. Scale bars, 500 μm and 100 μm. (B, C) Quantification of vascular progression (B) and vascular density (C) of P7 control (Ctrl) and Emc1 iECKO/iECKO mouse retinae. Error bars, standard deviations (SDs). Student's t -test ( n ≥ 7), ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. (D) Representative immunofluorescence images of P6 Ctrl and Emc1 iECKO/iECKO mouse retinae stained with IB4. Red dots indicate sprouts at the angiogenic front. Scale bars, 50 μm. (E) Quantification of the numbers of sprouts per unit of front length (mm). Error bars, SDs. Student's t -test ( n = 5), ∗∗ P < 0.01. (F) Representative immunofluorescence image of superficial and deep IB4-staining retinae of P9 Ctrl and Emc1 iECKO/iECKO mice. Scale bars, 50 μm. (G) Representative overview and high-magnification images of P6 Ctrl and Emc1 iECKO/iECKO mouse retinae co-stained with IB4, EdU, and ERG. Dotted boxes indicate magnified areas. Scale bars, 20 μm and 200 μm. (H) Quantification of the percentage of EdU+/ERG + cells to total ERG + cells per field. Error bars, SDs. Student's t -test ( n = 9), ∗∗∗∗ P < 0.0001. (I) Representative immunofluorescence images of P6 Ctrl and Emc1 iECKO/iECKO mouse retinae co-stained with IB4 and Ter119. White arrows indicate leakage of erythrocytes. Scale bars, 200 μm.

Article Snippet: Mice carrying loxp sites flanking exons 5 and 6 of Emc1 (Cyagen Biosciences, Suzhou, China; https://www.cyagen.com/cn/zh-cn/sperm-bank-cko/230866 ) generated using the CRISPR/Cas9 system in C57BL/6J genetic background were bred into Pdgfb-iCre-ER transgenic mice line, , The offspring animals were then bred with Emc1 loxp/loxp homozygous mice to produce Emc1 loxp/loxp Pdgfb-iCre-ER mice (hereafter named Emc1 iECKO/iECKO ).

Techniques: Control, Staining, Immunofluorescence

Journal: Genes & Diseases

Article Title: Defective EMC1 drives abnormal retinal angiogenesis via Wnt/β-catenin signaling and may be associated with the pathogenesis of familial exudative vitreoretinopathy

doi: 10.1016/j.gendis.2022.10.003

Figure Lengend Snippet: EMC1 regulates Wnt signaling through FZD4 receptor. (A) Principal component analysis for Ctrl and EMC1 KD (knockdown) HRECs. (B) Volcano plot of differentially expressed genes (DEGs) in Ctrl and EMC1 KD HRECs. The dash lines indicate the threshold of P = 0.05 and |log 2 FC| = 0.585. The significantly down-regulated and up-regulated genes are shown as blue and yellow dots, respectively. Genes with no significant alterations are shown as green dots. (C) Pathway classification of DEGs using the PANTHER classification system online. (D) Heatmap of the expression fold changes of several Wnt-targeted genes in Ctrl and EMC1 KD HRECs as determined by RNA-seq analysis. (E) RT-qPCR quantification of the relative mRNA levels of EMC1, MYC, CCND1, SLC2A1, DKK1, CTGF, SOX17, CD44, and PLVAP in Ctrl and EMC1 KD HRECs. Error bars, SDs. Student's t -test ( n = 3), ∗∗∗∗ P < 0.0001. (F) Relative luciferase activity in Ctrl and EMC1 KD HEK 293STF cells transfected with pGL4- Renilla as an internal control. Error bars, SDs. Student's t -test ( n = 6), ∗∗∗∗ P < 0.0001. (G – M) Western blot and quantification analysis of the expression levels of FLAG-FZD4, HA-LRP5, p-GSK3β, β-catenin, CyclinD1, and c-Myc in Ctrl and EMC1 KD HEK 293T cells co-transfected with FLAG-tagged FZD4, HA-tagged LRP5, and GFP. GFP and β-actin were used as internal controls for over-expressed proteins and endogenous proteins (unless otherwise noted), respectively. GSK3β was used as an internal control for p-GSK3β (unless otherwise noted). Error bars, SDs. Student's t -test ( n = 3), ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001. ns, not significant.

Article Snippet: Mice carrying loxp sites flanking exons 5 and 6 of Emc1 (Cyagen Biosciences, Suzhou, China; https://www.cyagen.com/cn/zh-cn/sperm-bank-cko/230866 ) generated using the CRISPR/Cas9 system in C57BL/6J genetic background were bred into Pdgfb-iCre-ER transgenic mice line, , The offspring animals were then bred with Emc1 loxp/loxp homozygous mice to produce Emc1 loxp/loxp Pdgfb-iCre-ER mice (hereafter named Emc1 iECKO/iECKO ).

Techniques: Knockdown, Expressing, RNA Sequencing, Quantitative RT-PCR, Luciferase, Activity Assay, Transfection, Control, Western Blot

Journal: Genes & Diseases

Article Title: Defective EMC1 drives abnormal retinal angiogenesis via Wnt/β-catenin signaling and may be associated with the pathogenesis of familial exudative vitreoretinopathy

doi: 10.1016/j.gendis.2022.10.003

Figure Lengend Snippet: EMC1 -depletion in HRECs results in compromised Wnt signaling and cell proliferation. (A – F) Western blot and quantification analysis of the expression levels of CyclinD1, c-Myc, p-GSK3β, FZD4, and β-catenin in Ctrl and EMC1 KD HRECs. Error bars, SDs. Student's t -test ( n = 3), ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. (G) RT-qPCR quantification of the relative FZD4 mRNA level in Ctrl and EMC1 KD HRECs. Error bars, SDs. Student's t -test ( n = 3). ns, not significant. (H) Representative immunofluorescence images of Ctrl and EMC1 KD HRECs labeled with EdU, β-catenin, or CyclinD1 and DAPI (blue). Scale bars, 20 μm (EdU and β-catenin) and 10 μm (CyclinD1). (I–K) Quantification of the percentage of EdU + nuclei (I) , and relative levels of CyclinD1 (J) and β-catenin (K) in Ctrl and EMC1 KD HRECs. Error bars, SDs. Student's t -test ( n ≥ 6), ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: Mice carrying loxp sites flanking exons 5 and 6 of Emc1 (Cyagen Biosciences, Suzhou, China; https://www.cyagen.com/cn/zh-cn/sperm-bank-cko/230866 ) generated using the CRISPR/Cas9 system in C57BL/6J genetic background were bred into Pdgfb-iCre-ER transgenic mice line, , The offspring animals were then bred with Emc1 loxp/loxp homozygous mice to produce Emc1 loxp/loxp Pdgfb-iCre-ER mice (hereafter named Emc1 iECKO/iECKO ).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Immunofluorescence, Labeling

Journal: Genes & Diseases

Article Title: Defective EMC1 drives abnormal retinal angiogenesis via Wnt/β-catenin signaling and may be associated with the pathogenesis of familial exudative vitreoretinopathy

doi: 10.1016/j.gendis.2022.10.003

Figure Lengend Snippet: LiCl treatment restores Wnt signaling activity and proliferation of EMC1 KD HRECs. (A) Relative luciferase activity in Ctrl, EMC1 KD, and EMC1 KD HEK 293STF cells treated with LiCl. pGL4- Renilla plasmid was transfected as an internal control. Error bars, SDs. The P -values are from multiple comparisons in one-way ANOVA with Tukey's multiple comparison test ( n = 6); ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001. (B – G) Western blot and quantification analysis of the expression levels of CyclinD1, c-Myc, p-GSK3β, β-catenin, and FZD4 in Ctrl and EMC1 KD HRECs treated with LiCl or NaCl. Error bars, SDs. The P -values are from multiple comparisons in two-way ANOVA with Sidak's multiple comparison test ( n = 3); ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. ns, not significant. (H) Representative immunofluorescence images of EMC1 KD HRECs treated with NaCl or LiCl and labeled with EdU, β-catenin, or CyclinD1 and DAPI (blue). Scale bars, 20 μm (EdU and β-catenin) and 10 μm (CyclinD1). (I–K) Quantification of the percentage of EdU + nuclei (I) , and relative levels of β-catenin (J) and CyclinD1 (K) in EMC1 KD HRECs treated with NaCl or LiCl. Error bars, SDs. Student's t -test ( n ≥ 6), ∗∗∗∗ P < 0.0001.

Article Snippet: Mice carrying loxp sites flanking exons 5 and 6 of Emc1 (Cyagen Biosciences, Suzhou, China; https://www.cyagen.com/cn/zh-cn/sperm-bank-cko/230866 ) generated using the CRISPR/Cas9 system in C57BL/6J genetic background were bred into Pdgfb-iCre-ER transgenic mice line, , The offspring animals were then bred with Emc1 loxp/loxp homozygous mice to produce Emc1 loxp/loxp Pdgfb-iCre-ER mice (hereafter named Emc1 iECKO/iECKO ).

Techniques: Activity Assay, Luciferase, Plasmid Preparation, Transfection, Control, Comparison, Western Blot, Expressing, Immunofluorescence, Labeling

Journal: Genes & Diseases

Article Title: Defective EMC1 drives abnormal retinal angiogenesis via Wnt/β-catenin signaling and may be associated with the pathogenesis of familial exudative vitreoretinopathy

doi: 10.1016/j.gendis.2022.10.003

Figure Lengend Snippet: Identification of a missense EMC1 variant in a FEVR-associated family. (A) FEVR pedigrees, Sanger sequencing analysis, and amino acids alignment in a Chinese FEVR-associated family. Patients are denoted in black. Black arrows indicate the proband of the family. (B) Fundus image of the proband's bilateral eyes. (C) Fundus fluorescein angiography of the proband's affected father.

Article Snippet: Mice carrying loxp sites flanking exons 5 and 6 of Emc1 (Cyagen Biosciences, Suzhou, China; https://www.cyagen.com/cn/zh-cn/sperm-bank-cko/230866 ) generated using the CRISPR/Cas9 system in C57BL/6J genetic background were bred into Pdgfb-iCre-ER transgenic mice line, , The offspring animals were then bred with Emc1 loxp/loxp homozygous mice to produce Emc1 loxp/loxp Pdgfb-iCre-ER mice (hereafter named Emc1 iECKO/iECKO ).

Techniques: Variant Assay, Sequencing

Journal: Genes & Diseases

Article Title: Defective EMC1 drives abnormal retinal angiogenesis via Wnt/β-catenin signaling and may be associated with the pathogenesis of familial exudative vitreoretinopathy

doi: 10.1016/j.gendis.2022.10.003

Figure Lengend Snippet: EMC1 I762V variant results in compromised Wnt signaling activity via reduced FZD4 expression. (A) Relative luciferase activity in HEK 293STF cells co-transfected with EMC1 (wild-type, wild-type/I762V, I762V variant, or vector) and pGL4- Renilla . pGL4- Renilla plasmid was transfected as an internal control. Error bars, SDs. The P -values are from multiple comparisons in one-way ANOVA with Tukey's multiple comparison test ( n = 4); ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. (B–F) RT-qPCR quantification of the relative mRNA levels of EMC1, CCND1, MYC, CTGF, and DKK1 in HEK 293T cells transfected with EMC1 (wild-type, I762V variant, or vector). Error bars, SDs. The P -values are from multiple comparisons in one-way ANOVA with Dunnett's multiple comparison test ( n = 3); ∗∗∗∗ P < 0.0001. (G – M) Western blot and quantification analysis of the expression levels of FLAG-EMC1, FZD4, p-GSK3β, β-catenin, c-Myc, and CyclinD1 in Ctrl and EMC1 KD HRECs. Error bars, SDs. The P -values are from multiple comparisons in one-way ANOVA with Tukey's multiple comparison test ( n = 3); ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. ns, not significant.

Article Snippet: Mice carrying loxp sites flanking exons 5 and 6 of Emc1 (Cyagen Biosciences, Suzhou, China; https://www.cyagen.com/cn/zh-cn/sperm-bank-cko/230866 ) generated using the CRISPR/Cas9 system in C57BL/6J genetic background were bred into Pdgfb-iCre-ER transgenic mice line, , The offspring animals were then bred with Emc1 loxp/loxp homozygous mice to produce Emc1 loxp/loxp Pdgfb-iCre-ER mice (hereafter named Emc1 iECKO/iECKO ).

Techniques: Variant Assay, Activity Assay, Expressing, Luciferase, Transfection, Plasmid Preparation, Control, Comparison, Quantitative RT-PCR, Western Blot

Journal: Infection and Immunity

Article Title: Involvement of endoplasmic reticulum and histone proteins in immunomodulation by TLR4-interacting SPA4 peptide against Escherichia coli

doi: 10.1128/iai.00311-23

Figure Lengend Snippet: SPA4 peptide induces co-localization of ER-proteins: calnexin and EMC1 with LAMP1 (phagolysosome marker) against challenge with heat-killed E. coli 19138 at 2.5 h. The schedule of infectious challenge and treatment is shown in (A). Representative images show the SPA4 peptide-induced intracellular co-localization of calnexin and EMC1 with LAMP1 against challenge with heat-killed E. coli (B, C). Representative images of unchallenged cells, treated with vehicle or SPA4 peptide, and stained for calnexin or EMC1 and LAMP1 are also shown (D, E). Bar charts show the percent number of cells showing co-localization of calnexin and EMC1 with LAMP1 (F-I). Results are from four experimental wells for each group set up on separate occasions. The p values are shown within the figure (t-test).

Article Snippet: The membrane with transferred proteins was immunoblotted with 1:500 or 1,000 diluted primary antibodies for calnexin (Abcam, Waltham, MA), EMC1 (Abclonal Technology, Woburn, MA), histone H2A (Cell Signaling Technology, Danvers, MA), and LAMP1 (Thermo Fisher Scientific, Waltham, MA) overnight and 1:500 or 1:1,000 diluted horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse antibody.

Techniques: Marker, Staining

Journal: Infection and Immunity

Article Title: Involvement of endoplasmic reticulum and histone proteins in immunomodulation by TLR4-interacting SPA4 peptide against Escherichia coli

doi: 10.1128/iai.00311-23

Figure Lengend Snippet: Assessment of expression of calnexin, EMC1, LAMP1, and histone H2A in cells challenged with heat-killed E. coli 19138 and treated with SPA4 peptide. The timeline of infectious challenge and SPA4 peptide treatment is shown in (A). The whole-cell lysate protein (2–5 µg) was separated and immunoblotted with antibodies specific to calnexin, EMC1, LAMP1, and histone H2A proteins. The membrane then was stripped and re-probed for β actin. Lanes contained whole-cell lysate protein from unchallenged, vehicle-treated control (1), heat-killed E. coli-challenged, vehicle-treated (2), and heat-killed E. coli-challenged, SPA4 peptide-treated (3) JAWS II dendritic cells. Representative immunoblots for each protein are shown within the figure (B, i–iv). The densitometric units for immunoreactive bands were normalized with those of β actin. The ratios of arbitrary densitometric units are shown as bar (Mean ± SEM) and are derived from three experiments performed on separate occasions (C, i–iv). The fold changes (calculated as normalized value for respective protein in experimental group per unchallenged, vehicle control group) are shown as bar (Mean ± SEM) in (D, i–iv). The p values are shown within each figure (one-way ANOVA).

Article Snippet: The membrane with transferred proteins was immunoblotted with 1:500 or 1,000 diluted primary antibodies for calnexin (Abcam, Waltham, MA), EMC1 (Abclonal Technology, Woburn, MA), histone H2A (Cell Signaling Technology, Danvers, MA), and LAMP1 (Thermo Fisher Scientific, Waltham, MA) overnight and 1:500 or 1:1,000 diluted horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse antibody.

Techniques: Expressing, Membrane, Western Blot, Derivative Assay

Journal: Nature Communications

Article Title: Homophilic ATP1A1 binding induces activin A secretion to promote EMT of tumor cells and myofibroblast activation

doi: 10.1038/s41467-022-30638-4

Figure Lengend Snippet: a ELISA analysis of activin A in fibroblasts generated from two distinct individuals (PSC and PSC-2) after direct contact with purified tumor membrane proteins (from BxPC-3, SU.86.86, MIA PaCa-2, or PANC-1 cells). Values were presented as mean ± SD ( n = 3). b Western blotting analysis of no cross-linking control and cross-linked membrane protein complexes with streptavidin-HRP under reducing condition. Since 2-mercaptoethanol broke disulfide bond on the cross-linker, a band (*) of biotin-labeled tumor membrane proteins from BxPC-3 and SU.86.86 cells were identified. c Five plasma membrane proteins were identified by mass spectrometry analysis. d ELISA analysis of activin A in mono-cultured or direct co-cultured PSCs with BxPC-3 cells stably expressing lentiviral-based LacZ shRNA , ATP1A1 shRNA Clone#1 , ATP1A2 shRNA , ATP1A3 shRNA , EMC1 shRNA Clone#1 , ITGB1 shRNA . Values were presented as mean ± SD ( n = 3). e ELISA analysis of activin A in fibroblasts co-cultured with 2% paraformaldehyde fixed BxPC-3 cells and treated with IgG (4 μg/ml), anti-ATP1A1-neutralizing antibody (2 and 4 μg/ml, 14418-1-AP, Proteintech), or anti-EMC1-neutralizing antibody (2 and 4 μg/ml, GTX119884, Genetex). Values were presented as mean ± SD ( n = 3). f Left panel, western blotting analysis of biotinylated fibroblast membrane proteins with streptavidin-HRP under reducing condition. A band (*) of biotin-labeled fibroblast membrane proteins which bound to ATP1A1 was identified. Right panel, re-probing the western blot membrane from left panel with anti-Flag antibody to confirm that the identified band (*) was not ATP1A1-Flag. g ATP1A1 was identified by mass spectrometry analysis. h ELISA analysis of activin A in mono-cultured and direct co-cultured BxPC-3 cells with PSCs stably expressing lentiviral-based LacZ shRNA and ATP1A1 shRNA Clone#1 . Values were presented as mean ± SD ( n = 3). i Immunofluorescence staining of ATP1A1 in fibroblasts (PSC-GFP) and cancer cells (BxPC-3-RFP and SU.86.86-RFP) measured by confocal microscope. Scale bar, 2 μm. j Live-cell confocal imaging of BxPC-3 cells stably expressing ATP1A1-GFP and PSCs stably expressing ATP1A1-RFP. Co-localization of ATP1A1 was compared between initiation and establishment of tumor-fibroblast contact within 15 mins. Scale bar, 5 μm. k , l Correlation of ATP1A1 and INHBA expression levels in PDAC patient specimen. k Representative images of ATP1A1 expression (brown color, IHC) and INHBA expression (activin A, pink, in situ hybridization, RNAscope). Scale bar, 25 μm. l Pearson’s correlation coefficient analysis of ATP1A1 and INHBA according to H score and tumor-adjacent INHBA + cells (%) ( n = 100, r = 0.52, p < 0.0001). ** p < 0.01, *** p < 0.001, **** p < 0.0001 (two-tailed t-test).

Article Snippet: Immunoblot analysis was performed with overnight incubation of anti-vimentin (1:1000; 5741, Cell Signaling), anti-claudin1 (1:1000; 4933, Cell Signaling), anti-Snail (1:1000; 3879, Cell Signaling), anti-Twist1 (1:1000; 711565, Invitrogen), anti-ZEB1 (1:1000; 3396, Cell Signaling), anti-ZEB2 (1:1000; 97885, Cell Signaling), anti-ATP1A1 (1:10000; 14418-1-AP, Proteintech), anti-ATP1A2 (1:1000; 16836-1-AP, Proteintech), anti-ATP1A3 (1:1000; 28030-1-AP, Proteintech), anti-EMC1 (1:1000; GTX119884, Genetex), anti-ITGB1 (1:1000; 12594-1-AP, Proteintech), anti-αSMA (1:1000; ab7817, abcam), anti-collagen1 (1:1000; ab34710, abcam), anti-Flag (1:1000; F3165, Sigma), anti-p-p65 (S536) (1:1000; 3033, Cell Signaling), and anti-p65 (1:1000; 3034, Cell Signaling). anti-GAPDH (1:10000; GTX627408, Genetex) was used as loading controls for total cell lysates or cytosolic fraction. anti-flotillin1 (1:1000; ab133497, abcam) was used as loading control for membrane fraction.

Techniques: Enzyme-linked Immunosorbent Assay, Generated, Purification, Western Blot, Labeling, Mass Spectrometry, Cell Culture, Stable Transfection, Expressing, shRNA, Immunofluorescence, Staining, Microscopy, Imaging, In Situ Hybridization, Two Tailed Test