Journal: Journal of Translational Autoimmunity

Article Title: A novel epigenetic regulation of JAM-A by EZH2-DNMT3A cascade contributes to T cell adhesion via the activation of Rap1a in lupus patients

doi: 10.1016/j.jtauto.2026.100362

Figure Lengend Snippet: Increased expression of JAM-A in SLE patient PBMCs and peripheral T cells. (A – D) FCM analysis of JAM-A + PBMCs and JAM-A + T cells. (A) JAM-A was stained with anti-human JAM-A-PE antibodies in the PBMCs of healthy individuals (red), new SLE patients (blue), SLE treated (green) and isotype controls (black). (B) Quantification of JAM-A + PBMCs from (A). (C) Double staining with both anti-human JAM-A-PE and anti-human CD3-FITC antibodies. (D) Quantification of JAM-A + T cells from (C), and the error bars indicate the SEM. Healthy, (healthy volunteers, n = 24); new SLE (SLE patients without clinical treatments, n = 9); SLE treated (SLE patients with clinical treatments, n = 15). (E – F) The correlations of EZH2 mRNA/β-actin and the JAM-A mRNA/β-actin in PBMCs ( E ) and T cells ( F ) from SLE patients. ∗ P < 0.05, ∗∗∗ P < 0.001.

Article Snippet: Cells were stimulated overnight with anti-CD3 and anti-CD28 antibodies and transfected with 100 nM EZH2 siRNAs (siG09121182926-1-5/si-h-EZH2_001 and siG09121182954-1-5/si-h-EZH2_002, RiboBio, China), JAM-A siRNAs (SC-43139, Santa Cruz Biotechnology, USA), and negative control siRNA (siN0000001-1-5 for EZH2, SC-37007 for JAM-A) in 6-well plates.

Techniques: Expressing, Staining, Double Staining

Journal: Journal of Translational Autoimmunity

Article Title: A novel epigenetic regulation of JAM-A by EZH2-DNMT3A cascade contributes to T cell adhesion via the activation of Rap1a in lupus patients

doi: 10.1016/j.jtauto.2026.100362

Figure Lengend Snippet: The crosstalk between EZH2/H3K27me3 and DNMT3A/JAM-A. (A, D) The protein expression in Jurkat cells or T cells treated with 5 μM GSK126 (A) or EZH2-siRNA (D), GAPDH or H3 protein as loading control. (B, E) The methylation frequency in JAM-A gene promoter of T cells under EZH2 inhibitor 5 μM GSK126 (B) or EZH2-siRNA treated (E). (C, F) Immunoprecipitated JAM-A with anti-DNMT3A or DNMT3A with anti-H3K27me3 ChIP-grade antibodies in T cells under 5 μM GSK126 (C) or EZH2-siRNA treated (F). The values were normalized to the chromatin levels with 1% input samples. (G, H) The negative correlations of EZH2 mRNA/β-actin and DNMT3A mRNA/β-actin (G) , DNMT3A mRNA/β-actin and JAM-A mRNA/β-actin (H) in T cells from SLE patients. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and error bars indicate the SEM.

Article Snippet: Cells were stimulated overnight with anti-CD3 and anti-CD28 antibodies and transfected with 100 nM EZH2 siRNAs (siG09121182926-1-5/si-h-EZH2_001 and siG09121182954-1-5/si-h-EZH2_002, RiboBio, China), JAM-A siRNAs (SC-43139, Santa Cruz Biotechnology, USA), and negative control siRNA (siN0000001-1-5 for EZH2, SC-37007 for JAM-A) in 6-well plates.

Techniques: Expressing, Control, Methylation, Immunoprecipitation

Journal: Journal of Translational Autoimmunity

Article Title: A novel epigenetic regulation of JAM-A by EZH2-DNMT3A cascade contributes to T cell adhesion via the activation of Rap1a in lupus patients

doi: 10.1016/j.jtauto.2026.100362

Figure Lengend Snippet: Inhibition of EZH2 and JAM-A suppresses β1 integrin-mediated T cells and Jurkat cells adhering to ECM. ( A ) The adhesion capacity in T cells from Healthy and SLE patients in vitro . ( B-C ) The adhesion capacity of T cells isolated from healthy or Jurkat cells blocked by 5 μM GSK126 (B) or EZH2-siRNA (C) in vitro . ( D ) Compared the adhesion capacity of T cells from SLE patients, or Jurkat cells with/out neutralizing anti-JAM-A, 1 μg/ml J10.4. (E) Protein expression levels in Jurkat cells overexpressing or knockdown of JAM-A. (F) The adhesion capacity of Jurkat cells with JAM-A regulation. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗ P < 0.001, and error bars indicate the SEM.

Article Snippet: Cells were stimulated overnight with anti-CD3 and anti-CD28 antibodies and transfected with 100 nM EZH2 siRNAs (siG09121182926-1-5/si-h-EZH2_001 and siG09121182954-1-5/si-h-EZH2_002, RiboBio, China), JAM-A siRNAs (SC-43139, Santa Cruz Biotechnology, USA), and negative control siRNA (siN0000001-1-5 for EZH2, SC-37007 for JAM-A) in 6-well plates.

Techniques: Inhibition, In Vitro, Isolation, Expressing, Knockdown

Journal: Journal of Translational Autoimmunity

Article Title: A novel epigenetic regulation of JAM-A by EZH2-DNMT3A cascade contributes to T cell adhesion via the activation of Rap1a in lupus patients

doi: 10.1016/j.jtauto.2026.100362

Figure Lengend Snippet: Inhibition of EZH2 with GSK126 ameliorates lupus-like disease phenotypes in MRL/ lpr mice. (A) Eight-week-old female MRL/ lpr mice were adaptively fed for 2 weeks (wks, n = 15) in the SPF animal room. Mice were randomly divided into a vehicle group (n = 8) and a GSK126-treated group (n = 7) and were treated with an intraperitoneal (IP) injection of β-SEB (20%) or GSK126 (50 mg/kg) every other day for one month. (B) Survival curves of the vehicle and treated group. (C) Anti-dsDNA antibody levels in the plasma. (D) The mRNA expression of IL-10 and TGF-β in splenomegaly. (E) Photomicrographic representation of renal damage (arrow). (F) The percentage plot of glomerulus damage. (G) The Representative histological images of immunohistochemical staining of JAM-A and β1-integrin antibody. (H) The quantification of positive staining areas was quantified by Saiviewer software. (I) The protein expression of EZH2, DNMT3A, JAM-A, Rap1a, β1 integrin and H3K27me3 in splenocytes detected by western blotting. (J) A total of 7 mice per group were analyzed for densitometry by image J software from (I). The symbols represent individual mice, and error bars indicate the SEM. ∗P < 0.05, ∗∗∗P < 0.001, ∗∗∗P < 0.001.

Article Snippet: Cells were stimulated overnight with anti-CD3 and anti-CD28 antibodies and transfected with 100 nM EZH2 siRNAs (siG09121182926-1-5/si-h-EZH2_001 and siG09121182954-1-5/si-h-EZH2_002, RiboBio, China), JAM-A siRNAs (SC-43139, Santa Cruz Biotechnology, USA), and negative control siRNA (siN0000001-1-5 for EZH2, SC-37007 for JAM-A) in 6-well plates.

Techniques: Inhibition, Injection, Clinical Proteomics, Expressing, Immunohistochemical staining, Staining, Software, Western Blot

Journal: Journal of Translational Autoimmunity

Article Title: A novel epigenetic regulation of JAM-A by EZH2-DNMT3A cascade contributes to T cell adhesion via the activation of Rap1a in lupus patients

doi: 10.1016/j.jtauto.2026.100362

Figure Lengend Snippet: Proposed schematic model of the EZH2/miR-26a-5p signaling axis involved in the development of SLE. Increased EZH2 expression is mediated by miR-26a-5p, which in turn is epigenetically repressed by EZH2-mediated H3K27me3 trimethylation within the miR-26a-5p promoter, thus forming a vicious cycle. Crosstalk between H3K27me3 and CpG island methylation mediated by DNMT3A within the JAM-A promoter, resulted in increased expression of JAM-A. Increased expression of JAM-A up-regulated the expression of Rap1a, a regulator of β1-integrin, which is functionally relevant to T cell adhesion. Combined with the increased transcriptional level of Rap1a mediated by miR-26a-5p, ultimately, Rap1a led to increased expression of β1-integrin, which is involved in increasing T cell adhesion.

Article Snippet: Cells were stimulated overnight with anti-CD3 and anti-CD28 antibodies and transfected with 100 nM EZH2 siRNAs (siG09121182926-1-5/si-h-EZH2_001 and siG09121182954-1-5/si-h-EZH2_002, RiboBio, China), JAM-A siRNAs (SC-43139, Santa Cruz Biotechnology, USA), and negative control siRNA (siN0000001-1-5 for EZH2, SC-37007 for JAM-A) in 6-well plates.

Techniques: Expressing, Methylation

Journal: Journal of Advanced Research

Article Title: JAM-C prevents ocular fibrosis by suppressing the TAZ/KLF6 pathway

doi: 10.1016/j.jare.2025.05.037

Figure Lengend Snippet: JAM-C inhibits TAZ activation in RPE. (A-E) Western blot showing the expression of JAM-C, TAZ, p-TAZ and YAP after JAM-C knockdown in RPE cells. n = 3. (F-H) Western blot analysis for TAZ after JAM-C knockdown and treatment with 25 µg/ml Cycloheximide (CHX) in RPE cells. n = 3. (I) Co-immunoprecipitation (co-IP) using anti-JAM-C antibody or IgG followed by Western blot showing the interaction of JAM-C, TAZ and YAP in RPE cells. n = 3. (J) Schematic representation of full-length JAM-C and its ΔCyto truncate. (K) Results of co-immunoprecipitation (co-IP) followed by Western blot showing the interaction between Flag-tagged JAM-C FL but not ΔCyto and TAZ. n = 3. (L-N) Western blot showing the subcellular portion of TAZ in RPE cells after JAM-C knockdown. GAPDH and HISTONE 3 were used as cytoplasmic or nuclear controls respectively. n = 3. (O-P) Immunostaining of JAM-C (red) and TAZ (green) in RPE cells after JAM-C knockdown. Nuclei are stained by DAPI. Scale bar: 20 μm. Quantification of the percentage of TAZ + cells in the nucleus are shown in P, n = 4. Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA in G, H, M−N and Student’s t -test in B-E, and P. ** p < 0.01, *** p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The cells were then blocked using 5 % bovine serum albumin (BSA, B2064, Sigma-Aldrich) at room temperature for 1 h, and incubated overnight at 4 °C with the indicated primary antibodies, including anti-JAM-C (AF1189, R&D Systems) and anti-fibronectin (F3648, Sigma-Aldrich) and anti-TAZ (HPA007415 Sigma-Aldrich).

Techniques: Activation Assay, Western Blot, Expressing, Knockdown, Immunoprecipitation, Co-Immunoprecipitation Assay, Immunostaining, Staining

Journal: Journal of Advanced Research

Article Title: JAM-C prevents ocular fibrosis by suppressing the TAZ/KLF6 pathway

doi: 10.1016/j.jare.2025.05.037

Figure Lengend Snippet: Genetic deletion of Jam-c exacerbates ocular fibrosis. (A) ELISA results showing lower levels of JAM-C in the vitreous humor of PVR patients. n = 9 for CTRL and n = 13 for PVR. (B-F) RPE cells were treated with TGF-β2 (2 and 4 ng/ml) for 48 h and subjected to Western blot for indicated proteins. n = 3. (G) Diagram showing the generation of the PVR mouse model in Jam-c RPE cko and CTRL mice. (H-J) Western blot showing the protein levels of JAM-C and RPE marker (RPE65) in the RPE-choroid complex of CTRL and Jam-c RPE cko mice. n = 3. (K-M) H&E staining showing epiretinal (black arrows) and subretinal (yellow arrows) membrane formation in Jam-c RPE cko and CTRL mice in a PVR model (K). RPE: retinal pigment epithelium, INL: inner nuclear layer, ONL: outer nuclear layer. Scale bar: 50 μm. The area of the membranes (K) are shown in L and M. n = 9 for CTRL and n = 8 for Jam-c RPE cko. (N-Q) DIC (differential interference contrast) and immunostaining of fibronectin in Jam-c RPE cko and CTRL mice in a PVR model (N). Green: fibronectin; Blue: DAPI. Red asterisks: misplaced melanosome; Orange arrows: subretinal membrane (SRM); Red arrows: epiretinal membrane (ERM); Yellow lines: RPE-choroid complex. Scale bar: 50 μm. The thickness of RPE-choroid complex and the fibronectin-positive membrane area (N) are shown in O-Q. n = 7 for CTRL and n = 6 for Jam-c RPE cko. Data are shown as mean ± SD. Statistical significance was assessed by one-way ANOVA in C-F, and Student’s t -test in A, I-J, L-M, and O-Q. * p < 0.05, ** p < 0.01, *** p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Cell lysates were incubated with JAM-C antibody (AF1189, R&D Systems, MN, USA), or IgG (AB-108-C, R&D Systems) at 4°C overnight, followed by Protein A/G-Sepharose (sc-2003, Santa Cruz, USA) incubation for 1.5 h at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Marker, Staining, Membrane, Immunostaining

Journal: Journal of Advanced Research

Article Title: JAM-C prevents ocular fibrosis by suppressing the TAZ/KLF6 pathway

doi: 10.1016/j.jare.2025.05.037

Figure Lengend Snippet: JAM-C knockdown results in RPE EMT. (A) qRT-PCR results showing the knockdown efficiency of JAM-C by two different siRNA oligos in RPE cells. n = 4. (B-F) Western blot showing the protein levels of JAM-C, mesenchymal markers (fibronectin, N-cadherin) and the epithelial marker E-cadherin in ARPE-19 cells with JAM-C knockdown. n = 3. (G-L) Western blot showing the protein levels of JAM-C, mesenchymal markers (fibronectin, vimentin, N-cadherin) and the epithelial marker E-cadherin in primary human RPE cells with JAM-C knockdown. n = 3. (M−N) Immunostaining of fibronectin in RPE cells with JAM-C knockdown (M). Nuclei are stained by DAPI. Scale bar: 10 μm. Quantification of fibronectin staining intensity is shown in N. n = 6. (O) CCK8 assay results showing the cell proliferation status after JAM-C knockdown in RPE cells at 72 h. n = 6. (P-Q) Wound healing assay results showing the migration potential of RPE cells after JAM-C knockdown (P). Scale bar: 200 μm. Quantification of wound closure areas are shown in Q. n = 5. (R-S) Gel contraction assay results showing the cell contraction ability after JAM-C knockdown (R). Quantification of the relative remaining areas of the gels are shown in S. n = 4. Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA in A, C-F, and Student’s t -test in H-L, N, O, Q and S. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cell lysates were incubated with JAM-C antibody (AF1189, R&D Systems, MN, USA), or IgG (AB-108-C, R&D Systems) at 4°C overnight, followed by Protein A/G-Sepharose (sc-2003, Santa Cruz, USA) incubation for 1.5 h at 4 °C.

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Marker, Immunostaining, Staining, CCK-8 Assay, Wound Healing Assay, Migration, Collagen Gel Contraction Assay

Journal: Journal of Advanced Research

Article Title: JAM-C prevents ocular fibrosis by suppressing the TAZ/KLF6 pathway

doi: 10.1016/j.jare.2025.05.037

Figure Lengend Snippet: JAM-C inhibits TAZ activation in RPE. (A-E) Western blot showing the expression of JAM-C, TAZ, p-TAZ and YAP after JAM-C knockdown in RPE cells. n = 3. (F-H) Western blot analysis for TAZ after JAM-C knockdown and treatment with 25 µg/ml Cycloheximide (CHX) in RPE cells. n = 3. (I) Co-immunoprecipitation (co-IP) using anti-JAM-C antibody or IgG followed by Western blot showing the interaction of JAM-C, TAZ and YAP in RPE cells. n = 3. (J) Schematic representation of full-length JAM-C and its ΔCyto truncate. (K) Results of co-immunoprecipitation (co-IP) followed by Western blot showing the interaction between Flag-tagged JAM-C FL but not ΔCyto and TAZ. n = 3. (L-N) Western blot showing the subcellular portion of TAZ in RPE cells after JAM-C knockdown. GAPDH and HISTONE 3 were used as cytoplasmic or nuclear controls respectively. n = 3. (O-P) Immunostaining of JAM-C (red) and TAZ (green) in RPE cells after JAM-C knockdown. Nuclei are stained by DAPI. Scale bar: 20 μm. Quantification of the percentage of TAZ + cells in the nucleus are shown in P, n = 4. Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA in G, H, M−N and Student’s t -test in B-E, and P. ** p < 0.01, *** p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Cell lysates were incubated with JAM-C antibody (AF1189, R&D Systems, MN, USA), or IgG (AB-108-C, R&D Systems) at 4°C overnight, followed by Protein A/G-Sepharose (sc-2003, Santa Cruz, USA) incubation for 1.5 h at 4 °C.

Techniques: Activation Assay, Western Blot, Expressing, Knockdown, Immunoprecipitation, Co-Immunoprecipitation Assay, Immunostaining, Staining

Journal: Journal of Advanced Research

Article Title: JAM-C prevents ocular fibrosis by suppressing the TAZ/KLF6 pathway

doi: 10.1016/j.jare.2025.05.037

Figure Lengend Snippet: TAZ increases KLF6′s expression and pro-EMT function. (A) Volcano plot showing the up- and down-regulated genes in TAZ overexpressing RPE cells. (B) qRT-PCR showing KLF6 level in TAZ overexpressing RPE cells. n = 3. (C-E) Western blot showing the protein levels of TAZ and KLF6 after TAZ knockdown in primary human RPE cells. n = 3. (F) Dual luciferase reporter gene assay showing the luciferase activity of FN1 promoter after the overexpression of TAZ and/or KLF6 in RPE cells. n = 3. (G) ChIP-qPCR assay detected the enrichment of KLF6 at FN1 promoter region in RPE cells. n = 3. (H) qRT-PCR results showing the expression level of FN1 in RPE cells overexpressing KLF6 and/or TAZ. n = 3. (I) qRT-PCR result showing KLF6 mRNA level after knockdown of JAM-C and/or TAZ in RPE cells. n = 4. (J-N) Western blot showing the protein levels of JAM-C, TAZ, KLF6 and fibronectin after the knockdown of JAM-C and/or TAZ in RPE cells. n = 3. Data are presented as mean ± SD. Statistical significance was determined using Student’s t-test in B and one-way ANOVA in D-I, K-N. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cell lysates were incubated with JAM-C antibody (AF1189, R&D Systems, MN, USA), or IgG (AB-108-C, R&D Systems) at 4°C overnight, followed by Protein A/G-Sepharose (sc-2003, Santa Cruz, USA) incubation for 1.5 h at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Knockdown, Luciferase, Reporter Gene Assay, Activity Assay, Over Expression, ChIP-qPCR

Journal: Journal of Advanced Research

Article Title: JAM-C prevents ocular fibrosis by suppressing the TAZ/KLF6 pathway

doi: 10.1016/j.jare.2025.05.037

Figure Lengend Snippet: KLF6 mediates JAM-C depletion-induced RPE EMT and ocular fibrosis. (A-E) Western blot showing the protein levels of KLF6 and mesenchymal markers (fibronectin, N-cadherin, vimentin) after KLF6 knockdown in RPE cells. n = 3. (F) CCK8 assay showing the cell proliferation after KLF6 knockdown in RPE cells at 72 h. n = 4. (G-I) Western blot showing the protein levels of JAM-C and KLF6 after knockdown of JAM-C and/or KLF6 in RPE cells. n = 3. (J) CCK8 assay showing cell proliferation after knockdown of JAM-C and/or KLF6 in RPE cells. n = 4. (K-L) Fibronectin immunostaining in RPE cells with JAM-C and/or KLF6 knockdown (K). Nuclei are stained by DAPI. Scale bar: 10 μm. Quantification of fibronectin staining intensity is shown in L. n = 6. Data are presented as mean ± SD. Statistical significance was determined using Student’s t -test in B-F and one-way ANOVA in H, I, J and L. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cell lysates were incubated with JAM-C antibody (AF1189, R&D Systems, MN, USA), or IgG (AB-108-C, R&D Systems) at 4°C overnight, followed by Protein A/G-Sepharose (sc-2003, Santa Cruz, USA) incubation for 1.5 h at 4 °C.

Techniques: Western Blot, Knockdown, CCK-8 Assay, Immunostaining, Staining

Journal: Journal of Advanced Research

Article Title: JAM-C prevents ocular fibrosis by suppressing the TAZ/KLF6 pathway

doi: 10.1016/j.jare.2025.05.037

Figure Lengend Snippet: JAM-C overexpression alleviates ocular fibrosis. (A) Diagram showing the generation of the PVR model in WT mice with RPE-specific overexpression of JAM-C or CTRL. AAV, adeno-associated virus. Ad, adenovirus. (B-D) Western blot showing the protein levels of JAM-C and the RPE marker RPE65 in the RPE-choroid complex from mice with JAM-C OE in PVR models. n = 3. (E-H) DIC and immunostaining of fibronectin in the RPE-choroid complex from mice with JAM-C OE in PVR model (E). Green: fibronectin, Blue: DAPI. Red asterisks: displaced melanosome, orange arrows: subretinal membrane (SRM), red arrows: epiretinal membrane (ERM), yellow lines: thickness of RPE-choroid complex. Scale bar: 50 μm. Quantification of the thickness of RPE-choroid complex and the fibronectin-positive membrane areas in E are shown in F-H. n = 5 for each group. (I-K) Immunostaining of KLF6 in the retina of mice with JAM-C OE in a PVR model (I). Green: KLF6, Blue: DAPI, Red arrows: epiretinal membrane (ERM) Orange arrows: subretinal membrane (SRM). Scale bar: 50 μm. Quantification of the KLF6 positive areas in I are shown in J and K. n = 5 for each group. (L-N) Western blot showing the protein levels of KLF6 and the EMT marker fibronectin in the RPE-choroid complex of mice with JAM-C OE in PVR models. n = 3. Data are presented as mean ± SD. Statistical significance was determined using Student’s t -test in C-D, F-H, J-K, M−N. * p < 0.05, ** p < 0.01, *** p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Cell lysates were incubated with JAM-C antibody (AF1189, R&D Systems, MN, USA), or IgG (AB-108-C, R&D Systems) at 4°C overnight, followed by Protein A/G-Sepharose (sc-2003, Santa Cruz, USA) incubation for 1.5 h at 4 °C.

Techniques: Over Expression, Virus, Western Blot, Marker, Immunostaining, Membrane

Journal: Journal of Advanced Research

Article Title: JAM-C prevents ocular fibrosis by suppressing the TAZ/KLF6 pathway

doi: 10.1016/j.jare.2025.05.037

Figure Lengend Snippet: JAM-C prevents RPE EMT and ocular fibrosis by suppressing the TAZ/KLF6 axis. In wild-type mice, JAM-C reduces TAZ expression, stability and nuclear transportation to inhibit its EMT-promoting function to prevent EMT and fibrosis, thus maintaining normal RPE and retina. In Jam-c knockout mice, in the absence of JAM-C, TAZ expression, stability and nuclear translocation increases, leading to the upregulation of KLF6 and KLF6-induced expression of many EMT causing genes, resulting in RPE EMT and fibrosis.

Article Snippet: Cell lysates were incubated with JAM-C antibody (AF1189, R&D Systems, MN, USA), or IgG (AB-108-C, R&D Systems) at 4°C overnight, followed by Protein A/G-Sepharose (sc-2003, Santa Cruz, USA) incubation for 1.5 h at 4 °C.

Techniques: Expressing, Knock-Out, Translocation Assay