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mouse anti human integrin β6 antibody (Bio-Techne corporation)

Name: mouse anti human integrin β6 antibody
Brand: Bio-Techne corporation
Rating: 91 (7 reviews)

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Bio-Techne corporation mouse anti human integrin β6 antibody
Mouse Anti Human Integrin β6 Antibody, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse anti human integrin β6 antibody/product/Bio-Techne corporation
Average 91 stars, based on 7 article reviews

mouse anti human integrin β6 antibody - by Bioz Stars, 2026-03

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Figure 1. The αVβ3 integrin increases NgR2 expression and is associated with NgR2. (A) Immunoblotting analysis of the expression levels of the αVβ3 integrin and NgR2 (n = 3, left panels), and of the αVβ3 integrin, the NE markers chromogranin A (CHGA) and neuron-specific enolase (NSE; n = 1, middle panels), in C4-2B cells that exogenously express αVβ3 or their mock control cells. Right panel, immunoblotting analysis of the expression levels of the αVβ3 integrin, and NgR2 in PC3 cells in which the αVβ3 integrin expression was downregulated using three different siRNAs against ITGB3 (the gene responsible for the β3 integrin subunit). Non-silencing siRNA (NS) was used as control. Calnexin was used as loading control. (B) PC3 cell lysates were immunoprecipitated with Abs against the αVβ3 integrin, NgR2, the β1 integrin, the αVβ6 integrin or their isotype controls (left panel) and the αVβ3 integrin, NgR2, and the αVβ6 integrin (right panel). Immunoprecipitates were analyzed for NgR2, the β1 integrin, the αVβ3 integrin, and the αVβ6 integrin by immunoblotting (n = 2). Immunoblotting analyses were performed under non-reducing (left panel) and reducing conditions (right panel). NgR2 was detected using two different Abs (left panel).

Journal: Scientific reports

Article Title: The NOGO receptor NgR2, a novel αVβ3 integrin effector, induces neuroendocrine differentiation in prostate cancer.

doi: 10.1038/s41598-022-21711-5

Figure Lengend Snippet: Figure 1. The αVβ3 integrin increases NgR2 expression and is associated with NgR2. (A) Immunoblotting analysis of the expression levels of the αVβ3 integrin and NgR2 (n = 3, left panels), and of the αVβ3 integrin, the NE markers chromogranin A (CHGA) and neuron-specific enolase (NSE; n = 1, middle panels), in C4-2B cells that exogenously express αVβ3 or their mock control cells. Right panel, immunoblotting analysis of the expression levels of the αVβ3 integrin, and NgR2 in PC3 cells in which the αVβ3 integrin expression was downregulated using three different siRNAs against ITGB3 (the gene responsible for the β3 integrin subunit). Non-silencing siRNA (NS) was used as control. Calnexin was used as loading control. (B) PC3 cell lysates were immunoprecipitated with Abs against the αVβ3 integrin, NgR2, the β1 integrin, the αVβ6 integrin or their isotype controls (left panel) and the αVβ3 integrin, NgR2, and the αVβ6 integrin (right panel). Immunoprecipitates were analyzed for NgR2, the β1 integrin, the αVβ3 integrin, and the αVβ6 integrin by immunoblotting (n = 2). Immunoblotting analyses were performed under non-reducing (left panel) and reducing conditions (right panel). NgR2 was detected using two different Abs (left panel).

Article Snippet: The following antibodies (Abs) were used: for the immunoblotting (IB) analysis, rabbit monoclonal Abs against the αVβ3 integrin (13166S, Cell Signaling) and Aurora Kinase A (14475S, Cell Signaling), polyclonal goat Abs against the αVβ6 integrin (AF2389, R&D system) and NgR2 (AF2776, R&D system), rabbit polyclonal Abs against calnexin (CANX, sc11397, Santa Cruz), actin (a2066, Sigma), RhoA (sc-179, Santa Cruz), TSG101 (Abcam, ab30871), mouse monoclonal Abs against RhoA (sc-418, Santa Cruz), NSE (LS-C197136, LSBio), Kindlin-2 (MAB2617, Millipore), were also used.

Techniques: Expressing, Western Blot, Control, Immunoprecipitation

Figure 2. NgR2 expression is increased in NEPrCa patients, NE cell lines, and NEPrCa PDXs. (A) Left panel, mRNA levels for RTN4RL2, SYP (Synaptophysin), KLK3 (Kallikrein Related Peptidase 3), and AR (Androgen Receptor) reported as Transcripts Per Million were compared across multiple PrCa cell lines in the CCLE database50. (B) RNA sequencing analysis for RTN4RL2 of metastatic CRPrCa specimens acquired through rapid autopsy of 98 patients. Specimens are classified based on their levels of AR and NE markers. AR-positive NE-negative (ARpos_NEneg, n = 76), AR low NE-negative (ARlow_NEneg, n = 13), AR-positive NE-positive (ARpos_Nepos, n = 9), AR-negative NE-negative (ARneg-NEneg, n = 8), and AR-negative NE-positive (ARneg_NEprs, n = 11). Statistical analysis was performed using GraphPad Prism (CA, USA) and differences between two groups were compared using unpaired student’s t-test. (C) RNA sequencing analysis of RTN4RL2 expression for CRPrCa and NEPrCa tumors from cBioPortal (dataset Neuroendocrine Prostate Cancer, Multi-Institute20). Differential expression between the two groups is estimated by student’s t-test. (D) Representative IHC staining for the αVβ3 integrin or NgR2 of LuCaP PDX TMAs (n =37) is shown. IgG was used as negative control. The bar at the bottom right corner of each panel represents 20 μm. (E) Heat map of the histochemical score for SYP, NgR2, and the αVβ3 integrin of each LuCaP is shown. *P = 0.029; **P = 0.003. Spearman correlation was performed and r-values are reported. The histochemical score for the αVβ3 integrin and SYP has been previously reported13.

Journal: Scientific reports

Article Title: The NOGO receptor NgR2, a novel αVβ3 integrin effector, induces neuroendocrine differentiation in prostate cancer.

doi: 10.1038/s41598-022-21711-5

Figure Lengend Snippet: Figure 2. NgR2 expression is increased in NEPrCa patients, NE cell lines, and NEPrCa PDXs. (A) Left panel, mRNA levels for RTN4RL2, SYP (Synaptophysin), KLK3 (Kallikrein Related Peptidase 3), and AR (Androgen Receptor) reported as Transcripts Per Million were compared across multiple PrCa cell lines in the CCLE database50. (B) RNA sequencing analysis for RTN4RL2 of metastatic CRPrCa specimens acquired through rapid autopsy of 98 patients. Specimens are classified based on their levels of AR and NE markers. AR-positive NE-negative (ARpos_NEneg, n = 76), AR low NE-negative (ARlow_NEneg, n = 13), AR-positive NE-positive (ARpos_Nepos, n = 9), AR-negative NE-negative (ARneg-NEneg, n = 8), and AR-negative NE-positive (ARneg_NEprs, n = 11). Statistical analysis was performed using GraphPad Prism (CA, USA) and differences between two groups were compared using unpaired student’s t-test. (C) RNA sequencing analysis of RTN4RL2 expression for CRPrCa and NEPrCa tumors from cBioPortal (dataset Neuroendocrine Prostate Cancer, Multi-Institute20). Differential expression between the two groups is estimated by student’s t-test. (D) Representative IHC staining for the αVβ3 integrin or NgR2 of LuCaP PDX TMAs (n =37) is shown. IgG was used as negative control. The bar at the bottom right corner of each panel represents 20 μm. (E) Heat map of the histochemical score for SYP, NgR2, and the αVβ3 integrin of each LuCaP is shown. *P = 0.029; **P = 0.003. Spearman correlation was performed and r-values are reported. The histochemical score for the αVβ3 integrin and SYP has been previously reported13.

Techniques: Expressing, RNA Sequencing, Quantitative Proteomics, Immunohistochemistry, Negative Control

Figure 3. NgR2 expression is increased in NEPrCa TKO mouse tumors. (A, B) Representative immunohistochemical staining of the αVβ3 integrin, NgR2, and SYP of prostate tumors (top row) and lung metastasis (bottom row) from TKO mice (A, n = 5) and of primary tumors from SKO mice (B, n = 5). (C) Representative immunohistochemical staining of NgR2 of prostate samples from wild-type mice (n = 4). IgG was used as negative control (A–C). The bars at the bottom right corner of each panel represent 20 μm.

Journal: Scientific reports

Article Title: The NOGO receptor NgR2, a novel αVβ3 integrin effector, induces neuroendocrine differentiation in prostate cancer.

doi: 10.1038/s41598-022-21711-5

Figure Lengend Snippet: Figure 3. NgR2 expression is increased in NEPrCa TKO mouse tumors. (A, B) Representative immunohistochemical staining of the αVβ3 integrin, NgR2, and SYP of prostate tumors (top row) and lung metastasis (bottom row) from TKO mice (A, n = 5) and of primary tumors from SKO mice (B, n = 5). (C) Representative immunohistochemical staining of NgR2 of prostate samples from wild-type mice (n = 4). IgG was used as negative control (A–C). The bars at the bottom right corner of each panel represent 20 μm.

Techniques: Expressing, Immunohistochemical staining, Staining, Negative Control

Figure 4. NgR2 induces NE markers and RhoA in CRPrCa and NEPrCa cells in vitro. (A) Immunoblotting analysis of the expression levels of the αVβ3 integrin (on a 7.5% PAGE), NgR2 and Aurora Kinase A (AURKA, on a 7.5% PAGE), and neuron-specific enolase (NSE) and RhoA on a 12.5% PAGE in NCI-H660 cells in which NgR2 expression was downregulated using a pool of siRNAs against RTN4RL2 (NgR2). AURKA and NSE protein expression, measured using densitometric analysis, is reduced 11.22 and 8.4 fold, respectively, when compared to the non-silencing siRNA control (n = 4). (B) Left panel, immunoblotting analysis of the expression levels of NgR2 (on a 7.5% PAGE); RhoA and synaptophysin (SYP, on a 10% PAGE) in C4-2B cells that exogenously express αVβ3 in which NgR2 expression was downregulated using a pool of siRNA against RTN4RL2. Right panel, immunoblotting analysis of the expression levels of the αVβ3 integrin, AURKA, and NSE (on a 12.5% PAGE) in C4-2B cells that exogenously express the αVβ3 integrin and in which NgR2 expression was downregulated using a pool of siRNAs against RTN4RL2 (n = 3). AURKA and NSE protein expression, measured using densitometric analysis, is reduced 3.59 and 3.9 fold, respectively, when compared to the non-silencing siRNA control. (A,B) –, Oligofectamine; NS, non-silencing. (C) Immunoblotting analysis of the expression levels of NgR2 (on a 10% PAGE), the αVβ3 integrin, NSE, and RhoA (on a 10% PAGE), and Chromogranin A (CHGA, on a 10% PAGE) in DU145 cells that exogenously express NgR2 or their mock control cells (n = 2). Actin or CANX was used as loading control (A–C). All immunoblotting analyses were performed under reducing conditions.

Journal: Scientific reports

Article Title: The NOGO receptor NgR2, a novel αVβ3 integrin effector, induces neuroendocrine differentiation in prostate cancer.

doi: 10.1038/s41598-022-21711-5

Figure Lengend Snippet: Figure 4. NgR2 induces NE markers and RhoA in CRPrCa and NEPrCa cells in vitro. (A) Immunoblotting analysis of the expression levels of the αVβ3 integrin (on a 7.5% PAGE), NgR2 and Aurora Kinase A (AURKA, on a 7.5% PAGE), and neuron-specific enolase (NSE) and RhoA on a 12.5% PAGE in NCI-H660 cells in which NgR2 expression was downregulated using a pool of siRNAs against RTN4RL2 (NgR2). AURKA and NSE protein expression, measured using densitometric analysis, is reduced 11.22 and 8.4 fold, respectively, when compared to the non-silencing siRNA control (n = 4). (B) Left panel, immunoblotting analysis of the expression levels of NgR2 (on a 7.5% PAGE); RhoA and synaptophysin (SYP, on a 10% PAGE) in C4-2B cells that exogenously express αVβ3 in which NgR2 expression was downregulated using a pool of siRNA against RTN4RL2. Right panel, immunoblotting analysis of the expression levels of the αVβ3 integrin, AURKA, and NSE (on a 12.5% PAGE) in C4-2B cells that exogenously express the αVβ3 integrin and in which NgR2 expression was downregulated using a pool of siRNAs against RTN4RL2 (n = 3). AURKA and NSE protein expression, measured using densitometric analysis, is reduced 3.59 and 3.9 fold, respectively, when compared to the non-silencing siRNA control. (A,B) –, Oligofectamine; NS, non-silencing. (C) Immunoblotting analysis of the expression levels of NgR2 (on a 10% PAGE), the αVβ3 integrin, NSE, and RhoA (on a 10% PAGE), and Chromogranin A (CHGA, on a 10% PAGE) in DU145 cells that exogenously express NgR2 or their mock control cells (n = 2). Actin or CANX was used as loading control (A–C). All immunoblotting analyses were performed under reducing conditions.

Techniques: In Vitro, Western Blot, Expressing, Control

Figure 5. NgR2 increases the anchorage-independent growth of PC3 PrCa cells. (A) Immunoblotting analysis of the αVβ3 integrin, and NgR2 expression levels in different PC3 cell transfectants in which NgR2 is downregulated using shRNA against RTN4RL2 (NgR2). CANX was used as loading control. The immunoblotting analysis was performed under reducing conditions. (B) Left panels, representative images of the colonies formed by the PC3 cells containing shRNA against RTN4RL2 (NgR2), the scramble shRNA control, and the parental cells. Right panel, average colony area of the PC3 cells shown in (A) grown in 0.3% agar as described in the Materials and Methods section. Each condition has been replicated three times, and a total of nine fields per condition has been recorded. Values are reported as mean ± SEM. Significance was calculated by student’s t-test.

Journal: Scientific reports

Article Title: The NOGO receptor NgR2, a novel αVβ3 integrin effector, induces neuroendocrine differentiation in prostate cancer.

doi: 10.1038/s41598-022-21711-5

Figure Lengend Snippet: Figure 5. NgR2 increases the anchorage-independent growth of PC3 PrCa cells. (A) Immunoblotting analysis of the αVβ3 integrin, and NgR2 expression levels in different PC3 cell transfectants in which NgR2 is downregulated using shRNA against RTN4RL2 (NgR2). CANX was used as loading control. The immunoblotting analysis was performed under reducing conditions. (B) Left panels, representative images of the colonies formed by the PC3 cells containing shRNA against RTN4RL2 (NgR2), the scramble shRNA control, and the parental cells. Right panel, average colony area of the PC3 cells shown in (A) grown in 0.3% agar as described in the Materials and Methods section. Each condition has been replicated three times, and a total of nine fields per condition has been recorded. Values are reported as mean ± SEM. Significance was calculated by student’s t-test.

Techniques: Western Blot, Expressing, shRNA, Control

Figure 7. The αVβ3 integrin must be active to induce NgR2 in PrCa cells. (A) Immunoblotting analysis of the expression levels of Kindlin-2, NgR2, αVβ3, and RhoA in the lysates from PC3 cells in which Kindlin-2 expression was downregulated using shRNA targeting FERMT2 (the gene responsible for Kindlin-2). CANX was used as loading control. (B) Flow cytometry analysis of αVβ3 activation by its capacity to bind soluble Fg-Alexa Fluor 488 in PC3 cells transfected with a shRNA against FERMT2 or the non-targeting shRNA control (n = 2). Values are reported as Mean Fluorescence Intensity (MFI), and P-values were calculated by unpaired t-test using GraphPad Prism. (C) Immunoblotting analysis of the expression levels of the αVβ3 integrin and Kindlin-2 in C4-2B cells that express the αVβ3 integrin or their mock control. Actin was used as loading control. All immunoblotting analyses were performed under reducing conditions. n.s., non-significant; ***P < 0.001.

Journal: Scientific reports

Article Title: The NOGO receptor NgR2, a novel αVβ3 integrin effector, induces neuroendocrine differentiation in prostate cancer.

doi: 10.1038/s41598-022-21711-5

Figure Lengend Snippet: Figure 7. The αVβ3 integrin must be active to induce NgR2 in PrCa cells. (A) Immunoblotting analysis of the expression levels of Kindlin-2, NgR2, αVβ3, and RhoA in the lysates from PC3 cells in which Kindlin-2 expression was downregulated using shRNA targeting FERMT2 (the gene responsible for Kindlin-2). CANX was used as loading control. (B) Flow cytometry analysis of αVβ3 activation by its capacity to bind soluble Fg-Alexa Fluor 488 in PC3 cells transfected with a shRNA against FERMT2 or the non-targeting shRNA control (n = 2). Values are reported as Mean Fluorescence Intensity (MFI), and P-values were calculated by unpaired t-test using GraphPad Prism. (C) Immunoblotting analysis of the expression levels of the αVβ3 integrin and Kindlin-2 in C4-2B cells that express the αVβ3 integrin or their mock control. Actin was used as loading control. All immunoblotting analyses were performed under reducing conditions. n.s., non-significant; ***P < 0.001.

Techniques: Western Blot, Expressing, shRNA, Control, Flow Cytometry, Activation Assay, Transfection, Fluorescence

Figure 8. Schematic representation of the pathway described in this paper. (A) The αVβ3 integrin, together with its co-activator K2, induces increased levels of NgR2 expression in castrate-resistant prostate cancer cells. In turn, NgR2 regulates the levels of NE markers and stimulates anchorage-independent growth of these cells. NgR2 also upregulates the levels of RhoA that causes increased PrCa cell motility. These cells with increased NgR2 expression are likely to be NEPrCa cells. (B) The schematic drawing summarizes our proposed model.

Journal: Scientific reports

Article Title: The NOGO receptor NgR2, a novel αVβ3 integrin effector, induces neuroendocrine differentiation in prostate cancer.

doi: 10.1038/s41598-022-21711-5

Figure Lengend Snippet: Figure 8. Schematic representation of the pathway described in this paper. (A) The αVβ3 integrin, together with its co-activator K2, induces increased levels of NgR2 expression in castrate-resistant prostate cancer cells. In turn, NgR2 regulates the levels of NE markers and stimulates anchorage-independent growth of these cells. NgR2 also upregulates the levels of RhoA that causes increased PrCa cell motility. These cells with increased NgR2 expression are likely to be NEPrCa cells. (B) The schematic drawing summarizes our proposed model.

Techniques: Expressing

(A) Immunoblotting (IB) analysis of total cell lysates (TCL) from PC3 cells containing the β6 integrin subunit, PC3-WT not treated with siRNA (−), PC3 cells treated with non-silencing siRNA (NS), or the β6 integrin subunit siRNA (D2) (30 µg). TCL were separated using 12.5% SDS–PAGE. TCL were examined for the expression of the β6 integrin subunit (left panel, non-reducing SDS–PAGE), IFIT3 and STAT1 (right panel, reducing SDS–PAGE). TSG101 (left panel, non-reducing SDS–PAGE) and Actin (right panel, reducing SDS–PAGE) were used as protein loading controls. (B) IB analysis of TCL from PC3-WT cells and PC3-CRISPR clones devoid of the β6 integrin subunit, β6KO C5 and C7 (30 µg). TCL were separated using 12.5% SDS–PAGE. TCL were examined for the expression of the β6 integrin subunit (left panel, non-reducing SDS–PAGE), IFIT3 and STAT1 (right panel, reducing SDS–PAGE). The Actin membrane (right panel, reducing SDS–PAGE) was stripped to visualize IFIT3. Actin (left panel, non-reducing SDS–PAGE), and (right panel, reducing SDS–PAGE) was used as a protein loading control.

Journal: The Biochemical journal

Article Title: IFIT3 (Interferon Induced Protein with Tetratricopeptide Repeats 3) Modulates STAT1 Expression in small Extracellular Vesicles

doi: 10.1042/BCJ20210580

Figure Lengend Snippet: (A) Immunoblotting (IB) analysis of total cell lysates (TCL) from PC3 cells containing the β6 integrin subunit, PC3-WT not treated with siRNA (−), PC3 cells treated with non-silencing siRNA (NS), or the β6 integrin subunit siRNA (D2) (30 µg). TCL were separated using 12.5% SDS–PAGE. TCL were examined for the expression of the β6 integrin subunit (left panel, non-reducing SDS–PAGE), IFIT3 and STAT1 (right panel, reducing SDS–PAGE). TSG101 (left panel, non-reducing SDS–PAGE) and Actin (right panel, reducing SDS–PAGE) were used as protein loading controls. (B) IB analysis of TCL from PC3-WT cells and PC3-CRISPR clones devoid of the β6 integrin subunit, β6KO C5 and C7 (30 µg). TCL were separated using 12.5% SDS–PAGE. TCL were examined for the expression of the β6 integrin subunit (left panel, non-reducing SDS–PAGE), IFIT3 and STAT1 (right panel, reducing SDS–PAGE). The Actin membrane (right panel, reducing SDS–PAGE) was stripped to visualize IFIT3. Actin (left panel, non-reducing SDS–PAGE), and (right panel, reducing SDS–PAGE) was used as a protein loading control.

Article Snippet: The following Abs were used for IB analyses: mouse monoclonal Abs against the human β6 integrin subunit (6.2A1) [ 34 ], CD9 (Santa Cruz, sc-13118), CD63 (Abcam, ab8219), CD81 (Abcam, ab23505), STAT1 (Santa Cruz, sc-271661) and IFIT3 (Santa Cruz, sc-393512); rabbit polyclonal Abs against Actin (Sigma–Aldrich, A2066) and calnexin (Cell signaling, 2433S); rabbit monoclonal TSG101 (Abcam, ab125011); and goat-affinity purified polyclonal Abs against the β6 integrin subunit (R&D Systems, AF2389).

Techniques: Western Blot, SDS Page, Expressing, CRISPR, Clone Assay, Membrane, Control

$(A) IB analysis of sEV fractions (F1-F10) isolated via iodixanol density gradients and derived from PC3 wild-type (PC3-WT) cells containing the β6 integrin subunit, (PC3-WT). PC3-WT TCL (20 µg), PC3-WT (PC3-WT 100K) sEV lysate (10 µg) and PC3-WT sEV fraction lysates were separated using 12.5% SDS–PAGE. Comparable volumes of each sEV fraction were loaded (30 µl). The 10 consecutive sEV fractions have densities of 1.103, 1.117,1.134,1.151, 1.169, 1.186, 1.193, 1.193, 1.200, and 1.207 g/ml, respectively. Expression of the β6 integrin subunit, CD63, CD81 (left panel, non-reducing SDS–PAGE) as well as STAT1, IFIT3 and CD9 (right panel, reducing SDS–PAGE) was analyzed in sEV fractions 1 to 10. The STAT1 membrane (right panel, reducing SDS–PAGE) was stripped to visualize calnexin (CNX). CNX (right panel, reducing SDS–PAGE) in PC3-β6KO C7, PC3-WT TCL, PC3-WT 100K as well as 10 consecutive PC3-WT derived sEV fractions was analyzed. PC3-WT 100K lysate was the input for the density gradient. (B) IB analysis of PC3-β6KO C7 derived sEV fractions isolated via density gradients. Total lysates (TCL 20 µg) from PC3-WT cells containing the β6 integrin subunit, as well as the PC3-CRISPR clone devoid of the β6 integrin subunit, PC3-β6KO C7, and PC3-β6KO C7 sEV fraction lysates were separated using 12.5% SDS–PAGE. Comparable volumes of each sEV fraction were loaded (30 µl). The 10 consecutive sEV fractions have densities of 1.075, 1.099, 1.123, 1.134, 1.151, 1.165, 1.169, 1.182, 1.186, and 1.193 g/ml, respectively. Expression of the β6 integrin, CD63, CD81 (left panel, non-reducing SDS–PAGE), STAT1, IFIT3 and CD9 (right panel, reducing SDS–PAGE) was analyzed in sEV fractions 1 to 10. The STAT1 membrane (right panel, reducing SDS–PAGE) was stripped to visualize calnexin (CNX). CNX (right panel, reducing SDS–PAGE) in the PC3-CRISPR clone lacking the β6 integrin subunit, PC3-β6KO C7 TCL and PC3-β6KO C7 derived sEV fractions was analyzed. A and B, TCL = total cell lysates.$

Journal: The Biochemical journal

Article Title: IFIT3 (Interferon Induced Protein with Tetratricopeptide Repeats 3) Modulates STAT1 Expression in small Extracellular Vesicles

doi: 10.1042/BCJ20210580

Figure Lengend Snippet: (A) IB analysis of sEV fractions (F1-F10) isolated via iodixanol density gradients and derived from PC3 wild-type (PC3-WT) cells containing the β6 integrin subunit, (PC3-WT). PC3-WT TCL (20 µg), PC3-WT (PC3-WT 100K) sEV lysate (10 µg) and PC3-WT sEV fraction lysates were separated using 12.5% SDS–PAGE. Comparable volumes of each sEV fraction were loaded (30 µl). The 10 consecutive sEV fractions have densities of 1.103, 1.117,1.134,1.151, 1.169, 1.186, 1.193, 1.193, 1.200, and 1.207 g/ml, respectively. Expression of the β6 integrin subunit, CD63, CD81 (left panel, non-reducing SDS–PAGE) as well as STAT1, IFIT3 and CD9 (right panel, reducing SDS–PAGE) was analyzed in sEV fractions 1 to 10. The STAT1 membrane (right panel, reducing SDS–PAGE) was stripped to visualize calnexin (CNX). CNX (right panel, reducing SDS–PAGE) in PC3-β6KO C7, PC3-WT TCL, PC3-WT 100K as well as 10 consecutive PC3-WT derived sEV fractions was analyzed. PC3-WT 100K lysate was the input for the density gradient. (B) IB analysis of PC3-β6KO C7 derived sEV fractions isolated via density gradients. Total lysates (TCL 20 µg) from PC3-WT cells containing the β6 integrin subunit, as well as the PC3-CRISPR clone devoid of the β6 integrin subunit, PC3-β6KO C7, and PC3-β6KO C7 sEV fraction lysates were separated using 12.5% SDS–PAGE. Comparable volumes of each sEV fraction were loaded (30 µl). The 10 consecutive sEV fractions have densities of 1.075, 1.099, 1.123, 1.134, 1.151, 1.165, 1.169, 1.182, 1.186, and 1.193 g/ml, respectively. Expression of the β6 integrin, CD63, CD81 (left panel, non-reducing SDS–PAGE), STAT1, IFIT3 and CD9 (right panel, reducing SDS–PAGE) was analyzed in sEV fractions 1 to 10. The STAT1 membrane (right panel, reducing SDS–PAGE) was stripped to visualize calnexin (CNX). CNX (right panel, reducing SDS–PAGE) in the PC3-CRISPR clone lacking the β6 integrin subunit, PC3-β6KO C7 TCL and PC3-β6KO C7 derived sEV fractions was analyzed. A and B, TCL = total cell lysates.

Techniques: Isolation, Derivative Assay, SDS Page, Expressing, Membrane, CRISPR

$(A) Nanoparticle tracking analysis (NTA) measurement of size distribution and concentration of sEV fractions 2 to 5 derived from cells containing the β6 integrin subunit (PC3-WT). (B) NTA measurement of size distribution and concentration of sEV fractions 2 to 5 derived from the PC3-CRISPR clone devoid of the β6 integrin subunit, PC3-β6KO C7.$

Journal: The Biochemical journal

Article Title: IFIT3 (Interferon Induced Protein with Tetratricopeptide Repeats 3) Modulates STAT1 Expression in small Extracellular Vesicles

doi: 10.1042/BCJ20210580

Figure Lengend Snippet: (A) Nanoparticle tracking analysis (NTA) measurement of size distribution and concentration of sEV fractions 2 to 5 derived from cells containing the β6 integrin subunit (PC3-WT). (B) NTA measurement of size distribution and concentration of sEV fractions 2 to 5 derived from the PC3-CRISPR clone devoid of the β6 integrin subunit, PC3-β6KO C7.

Techniques: Concentration Assay, Derivative Assay, CRISPR

$(A) NTA measurement of size distribution and concentration of density gradient-isolated sEVs (pooled fractions 2 to 5) derived from cells containing the β6 integrin subunit, PC3-CRISPR control and the PC3-CRISPR clones devoid of the β6 integrin subunit, β6KO C5 and β6KO C7. (B) Transmission electron microscopy analysis of density gradient-isolated sEVs (pooled fractions 2 to 5) derived from PC3-CRISPR control and β6KO C5 cells. Scale bar = 100 nm.$

Journal: The Biochemical journal

Article Title: IFIT3 (Interferon Induced Protein with Tetratricopeptide Repeats 3) Modulates STAT1 Expression in small Extracellular Vesicles

doi: 10.1042/BCJ20210580

Figure Lengend Snippet: (A) NTA measurement of size distribution and concentration of density gradient-isolated sEVs (pooled fractions 2 to 5) derived from cells containing the β6 integrin subunit, PC3-CRISPR control and the PC3-CRISPR clones devoid of the β6 integrin subunit, β6KO C5 and β6KO C7. (B) Transmission electron microscopy analysis of density gradient-isolated sEVs (pooled fractions 2 to 5) derived from PC3-CRISPR control and β6KO C5 cells. Scale bar = 100 nm.

Techniques: Concentration Assay, Isolation, Derivative Assay, CRISPR, Control, Clone Assay, Transmission Assay, Electron Microscopy

$(A) IB analysis of PC3-WT and the PC3-CRISPR clones devoid of IFIT3: PC3-IFIT3KO C13 and PC3-IFIT3KO C15 TCL (total cell lysates) (30 µg). TCL were separated using 12.5% SDS–PAGE and examined for the expression of STAT1 and IFIT3 (reducing SDS–PAGE). The IFIT3 membrane (reducing SDS–PAGE) was stripped to visualize Actin. Actin (reducing SDS–PAGE) was used as a protein loading control. Dark and light exposures shown (Top and bottom, respectively). (B) IB analysis of cells expressing the β6 integrin subunit, PC3-CRISPR control cell-derived sEV fractions isolated via density gradients. PC3-CRISPR control TCL (20 µg), PC3-CRISPR control (CRISPR control 100K) sEV lysate (10 µg), and PC3-CRISPR control sEV fraction lysates were separated by using 12.5% SDS–PAGE. Comparable volumes of each sEV fraction were loaded (30 µl). The 10 consecutive sEV fractions have a density of 1.099, 1.113,1.123,1.137, 1.148, 1.162, 1.179, 1.186, 1.193, and 1.210 g/ml, respectively. Expression of the β6 integrin subunit, CD63, CD81 (left panel, non-reducing SDS–PAGE), STAT1, IFIT3, TSG101 and CD9 (right panel, reducing SDS–PAGE) was analyzed in sEV fractions 1 to 10. IFIT3 (right panel, reducing SDS–PAGE) and STAT1 membranes (right panel, reducing SDS–PAGE) were stripped to visualize TSG101 and calnexin (CNX), respectively. CNX expression (right panel, reducing SDS–PAGE) in PC3-CRISPR control TCL, CRISPR control 100K as well as 10 consecutive PC3-CRISPR control cell-derived sEV fractions is shown. CRISPR control 100K sEV lysate was used as input for the density gradient. (C) IB analysis of sEV fractions derived from a PC3-CRISPR clone devoid of IFIT3, designated as PC3-IFIT3KO C13, and isolated via density gradients. PC3-IFIT3KO C13 TCL (20 µg), PC3-IFIT3KO C13 (IFIT3KO C13 100K) sEV lysate (10 µg) and PC3-IFIT3KO C13 sEV fraction lysates were separated using 12.5% SDS–PAGE. Comparable volumes of each sEV fraction were loaded (30 µl). The 10 consecutive sEV fractions have a density of 1.106, 1.110,1.130,1.134, 1.155, 1.169, 1.175, 1.182, 1.182, and 1.189 g/ml, respectively. Expression of the β6 integrin subunit, CD63 and CD81 (left panel, non-reducing SDS–PAGE) is analyzed in sEV fractions 1 to 10. The β6 integrin subunit membrane (left panel, reducing SDS–PAGE) was stripped to visualize calnexin (CNX). CNX expression (left panel, non-reducing SDS–PAGE) in PC3-IFIT3KO C13 TCL, PC3-WT 100K, IFIT3 C13 100K and 10 consecutive PC3-IFIT3KO C13 cell-derived sEV fractions is shown. Expression of STAT1, TSG101 and CD9 (right panel, reducing SDS–PAGE) is analyzed. IFIT3KO C13 100K was the input for the density gradient.$

Journal: The Biochemical journal

Article Title: IFIT3 (Interferon Induced Protein with Tetratricopeptide Repeats 3) Modulates STAT1 Expression in small Extracellular Vesicles

doi: 10.1042/BCJ20210580

Figure Lengend Snippet: (A) IB analysis of PC3-WT and the PC3-CRISPR clones devoid of IFIT3: PC3-IFIT3KO C13 and PC3-IFIT3KO C15 TCL (total cell lysates) (30 µg). TCL were separated using 12.5% SDS–PAGE and examined for the expression of STAT1 and IFIT3 (reducing SDS–PAGE). The IFIT3 membrane (reducing SDS–PAGE) was stripped to visualize Actin. Actin (reducing SDS–PAGE) was used as a protein loading control. Dark and light exposures shown (Top and bottom, respectively). (B) IB analysis of cells expressing the β6 integrin subunit, PC3-CRISPR control cell-derived sEV fractions isolated via density gradients. PC3-CRISPR control TCL (20 µg), PC3-CRISPR control (CRISPR control 100K) sEV lysate (10 µg), and PC3-CRISPR control sEV fraction lysates were separated by using 12.5% SDS–PAGE. Comparable volumes of each sEV fraction were loaded (30 µl). The 10 consecutive sEV fractions have a density of 1.099, 1.113,1.123,1.137, 1.148, 1.162, 1.179, 1.186, 1.193, and 1.210 g/ml, respectively. Expression of the β6 integrin subunit, CD63, CD81 (left panel, non-reducing SDS–PAGE), STAT1, IFIT3, TSG101 and CD9 (right panel, reducing SDS–PAGE) was analyzed in sEV fractions 1 to 10. IFIT3 (right panel, reducing SDS–PAGE) and STAT1 membranes (right panel, reducing SDS–PAGE) were stripped to visualize TSG101 and calnexin (CNX), respectively. CNX expression (right panel, reducing SDS–PAGE) in PC3-CRISPR control TCL, CRISPR control 100K as well as 10 consecutive PC3-CRISPR control cell-derived sEV fractions is shown. CRISPR control 100K sEV lysate was used as input for the density gradient. (C) IB analysis of sEV fractions derived from a PC3-CRISPR clone devoid of IFIT3, designated as PC3-IFIT3KO C13, and isolated via density gradients. PC3-IFIT3KO C13 TCL (20 µg), PC3-IFIT3KO C13 (IFIT3KO C13 100K) sEV lysate (10 µg) and PC3-IFIT3KO C13 sEV fraction lysates were separated using 12.5% SDS–PAGE. Comparable volumes of each sEV fraction were loaded (30 µl). The 10 consecutive sEV fractions have a density of 1.106, 1.110,1.130,1.134, 1.155, 1.169, 1.175, 1.182, 1.182, and 1.189 g/ml, respectively. Expression of the β6 integrin subunit, CD63 and CD81 (left panel, non-reducing SDS–PAGE) is analyzed in sEV fractions 1 to 10. The β6 integrin subunit membrane (left panel, reducing SDS–PAGE) was stripped to visualize calnexin (CNX). CNX expression (left panel, non-reducing SDS–PAGE) in PC3-IFIT3KO C13 TCL, PC3-WT 100K, IFIT3 C13 100K and 10 consecutive PC3-IFIT3KO C13 cell-derived sEV fractions is shown. Expression of STAT1, TSG101 and CD9 (right panel, reducing SDS–PAGE) is analyzed. IFIT3KO C13 100K was the input for the density gradient.

Techniques: CRISPR, Clone Assay, SDS Page, Expressing, Membrane, Control, Derivative Assay, Isolation

$(A) IB analysis of PC3-CRISPR control cells and PC3-CRISPR clones devoid of STAT1, PC3-STAT1KO C21 as well as PC3-STAT1KO C1C5 TCL (total cell lysates) (30 µg). TCL were separated using 12.5% SDS–PAGE. TCL were examined for the expression of STAT1, IFIT3, and Actin (reducing SDS–PAGE). The IFIT3 membrane (reducing SDS–PAGE) was stripped to visualize Actin. Actin was used as a protein loading control. (B) NTA measurement of size distribution and concentration of PC3-STAT1KO C21 derived sEV fractions 2-5. (C) IB analysis of sEV fractions derived from a PC3-CRISPR clone lacking STAT1, designated as PC3-STAT1KO C21, and isolated via density gradients. PC3-β6KO C7, PC3-STAT1KO C21 TCL (20 µg), PC3-STAT1KO C21 (STAT1KO C21 100K) sEV lysate (10 µg) and PC3-STAT1KO C21 sEV fraction lysates were separated by using 12.5% SDS–PAGE. Comparable volumes of each sEV fraction were loaded (30 µl). The 10 consecutive sEV fractions have a density of 1.106, 1.123,1.127,1.144 1.165, 1.169,1.186, 1.193, 1.200, and 1.207 g/ml, respectively. Expression of STAT1, IFIT3, TSG101, CD9 and CD81 (reducing SDS–PAGE) was analyzed in sEV fractions 1 to 10. The IFIT3, STAT1 and CD9 membranes (reducing SDS–PAGE) were stripped to visualize TSG101, calnexin (CNX), and CD81 respectively. CNX expression (reducing SDS–PAGE) was analyzed in PC3-CRISPR clone devoid of the β6 integrin subunit, PC3-β6KO C7 as well as PC3-CRISPR clone lacking STAT1, PC3-STAT1KO C21 TCL, STAT1KO C21 100K and 10 consecutive PC3-STAT1KO C21 derived sEV fractions. PC3-STAT1KO C21 100K sEV lysate was used as input for the density gradient.$

Journal: The Biochemical journal

Article Title: IFIT3 (Interferon Induced Protein with Tetratricopeptide Repeats 3) Modulates STAT1 Expression in small Extracellular Vesicles

doi: 10.1042/BCJ20210580

Figure Lengend Snippet: (A) IB analysis of PC3-CRISPR control cells and PC3-CRISPR clones devoid of STAT1, PC3-STAT1KO C21 as well as PC3-STAT1KO C1C5 TCL (total cell lysates) (30 µg). TCL were separated using 12.5% SDS–PAGE. TCL were examined for the expression of STAT1, IFIT3, and Actin (reducing SDS–PAGE). The IFIT3 membrane (reducing SDS–PAGE) was stripped to visualize Actin. Actin was used as a protein loading control. (B) NTA measurement of size distribution and concentration of PC3-STAT1KO C21 derived sEV fractions 2-5. (C) IB analysis of sEV fractions derived from a PC3-CRISPR clone lacking STAT1, designated as PC3-STAT1KO C21, and isolated via density gradients. PC3-β6KO C7, PC3-STAT1KO C21 TCL (20 µg), PC3-STAT1KO C21 (STAT1KO C21 100K) sEV lysate (10 µg) and PC3-STAT1KO C21 sEV fraction lysates were separated by using 12.5% SDS–PAGE. Comparable volumes of each sEV fraction were loaded (30 µl). The 10 consecutive sEV fractions have a density of 1.106, 1.123,1.127,1.144 1.165, 1.169,1.186, 1.193, 1.200, and 1.207 g/ml, respectively. Expression of STAT1, IFIT3, TSG101, CD9 and CD81 (reducing SDS–PAGE) was analyzed in sEV fractions 1 to 10. The IFIT3, STAT1 and CD9 membranes (reducing SDS–PAGE) were stripped to visualize TSG101, calnexin (CNX), and CD81 respectively. CNX expression (reducing SDS–PAGE) was analyzed in PC3-CRISPR clone devoid of the β6 integrin subunit, PC3-β6KO C7 as well as PC3-CRISPR clone lacking STAT1, PC3-STAT1KO C21 TCL, STAT1KO C21 100K and 10 consecutive PC3-STAT1KO C21 derived sEV fractions. PC3-STAT1KO C21 100K sEV lysate was used as input for the density gradient.

Techniques: CRISPR, Control, Clone Assay, SDS Page, Expressing, Membrane, Concentration Assay, Derivative Assay, Isolation

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