primary antibodies against gramd4  (Proteintech)

Journal: Clinical and Translational Medicine

Article Title: GRAMD4 inhibits tumour metastasis by recruiting the E3 ligase ITCH to target TAK1 for degradation in hepatocellular carcinoma

doi: 10.1002/ctm2.635

Figure Lengend Snippet: GRAMD4 expression was downregulated in HCC and predicted poor clinical outcomes. (A) GRAMD4 mRNA expression in HCC tissues and adjacent normal samples extracted from GSE22058, GSE14520, GSE63898 and TCGA. (B) GRAMD4 CNV status in HCC from TCGA. (C) Representative immunohistochemical staining images of GRAMD4 in 110 paired HCC tumour tissues and adjacent non‐tumour tissues (Scale bar: 250 μm, 25 μm). (D) Statistical analysis of GRAMD4 expression according to the IHC assay in HCC tissues and matched adjacent tissues. (E) Univariate regression analysis of the relation between the GRAMD4 and clinicopathological characteristics regarding OS in the Tongji cohort. Overall survival (F) and disease‐free survival (G) of HCC patients with low or high tumour GRAMD4 scoring in the Tongji cohort. Overall survival (H) of HCC patients with low or high GRAMD4 expression in the TCGA cohort

Article Snippet: Primary antibodies against GRAMD4 (24299‐1‐AP for WB, immunohistochemistry (IHC), immunofluorescence (IF) and (IP) and ITCH (20920‐1‐AP for WB, IP) were purchased from Proteintech (Wuhan, Hubei, China); primary antibodies against TAK1 (#5206 for WB, IHC, IF and IP), ERK (#4695 for WB), phospho‐ERK (#4370 for WB), p38 ((#8690 for WB), phospho‐p38 (#9215 for WB), JNK (#9252 for WB), phospho‐JNK(#9255 for WB) and Myc‐tag (#2276 for WB) were from CST (Danvers, MA, USA); Mouse serum IgG (I5381 for IP control), Rabbit serum IgG (I5006 for IP control), primary antibodies against Flag‐tag (F1804 for WB, IP and IF) and HA‐tag (H6908 for WB, IP and IF) were from Sigma (St. Louis, MO, USA); HRP‐conjugated anti‐Rabbit IgG (#111‐035‐003) and HRP‐conjugated anti‐Mouse IgG (#115‐035‐003) secondary antibodies were used for WB analysis.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Clinical and Translational Medicine

Article Title: GRAMD4 inhibits tumour metastasis by recruiting the E3 ligase ITCH to target TAK1 for degradation in hepatocellular carcinoma

doi: 10.1002/ctm2.635

Figure Lengend Snippet: GRAMD4 suppresses metastasis of HCC in vitro and in vivo. (A) Trans‐well migration and invasion assays were performed using HLF‐Vector and HLF‐GRAMD4 stably transformed cells. (B) Trans‐well migration and invasion assays were performed with Hep3B‐Ctrl, Hep3B‐Sh1 and Hep3B‐Sh2 stably transfected cells. Wound healing assays were performed with HLF‐GRAMD4 (C) and Hep3B‐shGRAMD4 (D) stably transfected cells. Representative images are shown. Experiments were performed in triplicates and the data are shown as mean ± SD. Scale bar, 100 μm. Lung metastasis experiments were conducted in nude mice with HLF‐GRAMD4 (E, G) and Hep3B‐shGRAMD4 (F, H) stably transfected cells. Representative images of lung metastases and H&E staining of lung tissues are shown. The data represent mean ± SD. Statistical analysis was performed using Student's unpaired t ‐test in (A‐D). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Primary antibodies against GRAMD4 (24299‐1‐AP for WB, immunohistochemistry (IHC), immunofluorescence (IF) and (IP) and ITCH (20920‐1‐AP for WB, IP) were purchased from Proteintech (Wuhan, Hubei, China); primary antibodies against TAK1 (#5206 for WB, IHC, IF and IP), ERK (#4695 for WB), phospho‐ERK (#4370 for WB), p38 ((#8690 for WB), phospho‐p38 (#9215 for WB), JNK (#9252 for WB), phospho‐JNK(#9255 for WB) and Myc‐tag (#2276 for WB) were from CST (Danvers, MA, USA); Mouse serum IgG (I5381 for IP control), Rabbit serum IgG (I5006 for IP control), primary antibodies against Flag‐tag (F1804 for WB, IP and IF) and HA‐tag (H6908 for WB, IP and IF) were from Sigma (St. Louis, MO, USA); HRP‐conjugated anti‐Rabbit IgG (#111‐035‐003) and HRP‐conjugated anti‐Mouse IgG (#115‐035‐003) secondary antibodies were used for WB analysis.

Techniques: In Vitro, In Vivo, Migration, Plasmid Preparation, Stable Transfection, Transformation Assay, Transfection, Staining

Journal: Clinical and Translational Medicine

Article Title: GRAMD4 inhibits tumour metastasis by recruiting the E3 ligase ITCH to target TAK1 for degradation in hepatocellular carcinoma

doi: 10.1002/ctm2.635

Figure Lengend Snippet: GRAMD4 interacted and co‐localized with TAK1. (A) MS analysis of GRAMD4‐associated proteins. (B) Co‐IP analysis of the binding between exogenous GRAMD4‐Flag and TAK1‐HA in co‐transfected HEK293T cells. (C‐D) Co‐IP analysis of the binding between endogenous GRAMD4 and TAK1 in HLF and Hep3B cells. (E) HLF cells were fixed and stained with TAK1 antibody (Red) and GRAMD4 antibody (Green). Nuclei were counterstained with DAPI (blue). Scale bar: 20μm. (F) Schematic illustration showing the wild‐type and truncation mutants of GRAMD4. (G) Co‐IP analysis of the interaction between TAK1 and full‐length GRAMD4 or its truncation mutants in HEK293T cells co‐transfected with TAK1‐HA plasmid and GRAMD4‐Flag plasmid or GRAMD4‐Flag truncation mutant plasmids

Article Snippet: Primary antibodies against GRAMD4 (24299‐1‐AP for WB, immunohistochemistry (IHC), immunofluorescence (IF) and (IP) and ITCH (20920‐1‐AP for WB, IP) were purchased from Proteintech (Wuhan, Hubei, China); primary antibodies against TAK1 (#5206 for WB, IHC, IF and IP), ERK (#4695 for WB), phospho‐ERK (#4370 for WB), p38 ((#8690 for WB), phospho‐p38 (#9215 for WB), JNK (#9252 for WB), phospho‐JNK(#9255 for WB) and Myc‐tag (#2276 for WB) were from CST (Danvers, MA, USA); Mouse serum IgG (I5381 for IP control), Rabbit serum IgG (I5006 for IP control), primary antibodies against Flag‐tag (F1804 for WB, IP and IF) and HA‐tag (H6908 for WB, IP and IF) were from Sigma (St. Louis, MO, USA); HRP‐conjugated anti‐Rabbit IgG (#111‐035‐003) and HRP‐conjugated anti‐Mouse IgG (#115‐035‐003) secondary antibodies were used for WB analysis.

Techniques: Co-Immunoprecipitation Assay, Binding Assay, Transfection, Staining, Plasmid Preparation, Mutagenesis

Journal: Clinical and Translational Medicine

Article Title: GRAMD4 inhibits tumour metastasis by recruiting the E3 ligase ITCH to target TAK1 for degradation in hepatocellular carcinoma

doi: 10.1002/ctm2.635

Figure Lengend Snippet: GRAMD4 exerted its tumour suppressive effects by modulating the protein levels of TAK1. (A) Western blot analysis of TAK1, p‐JNK, p‐p38, p‐ERK and p‐p65 protein levels in HLF cells stably transfected with GRAMD4 plasmid or vector control. (B) Western blot analysis of TAK1, p‐JNK, p‐p38, p‐ERK, and p‐65 protein levels in Hep3B cells stably transfected with lentivirus expressing Sh‐Ctrl, GRAMD4‐Sh1, or GRAMD4‐Sh2. (C) Western blot analysis of TAK1, p‐JNK, p‐p38, p‐ERK and p‐65 protein levels in GRAMD4‐depleted Hep3B cells transiently transfected with si‐NC, TAK1‐si. (D, E) The expression levels of MMP‐1, MMP‐3, MMP‐9, MMP‐10 and MMP‐13 in the indicated cells were analysed by qRT‐PCR. Experiments were performed in triplicate and data are shown as mean ± SD. (F) Transwell migration and invasion assays were performed with stably transfected HLF‐vector and HLF‐GRAMD4 cells. (G, H) Lung metastasis experiments were conducted in nude mice with the indicated stably transfected cells. Representative images of lung metastases (G) and H&E staining of lung tissues (H) are shown. Statistical analysis was performed using Student's unpaired t ‐test in (F), and the Mann–Whitney U test in (G). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Primary antibodies against GRAMD4 (24299‐1‐AP for WB, immunohistochemistry (IHC), immunofluorescence (IF) and (IP) and ITCH (20920‐1‐AP for WB, IP) were purchased from Proteintech (Wuhan, Hubei, China); primary antibodies against TAK1 (#5206 for WB, IHC, IF and IP), ERK (#4695 for WB), phospho‐ERK (#4370 for WB), p38 ((#8690 for WB), phospho‐p38 (#9215 for WB), JNK (#9252 for WB), phospho‐JNK(#9255 for WB) and Myc‐tag (#2276 for WB) were from CST (Danvers, MA, USA); Mouse serum IgG (I5381 for IP control), Rabbit serum IgG (I5006 for IP control), primary antibodies against Flag‐tag (F1804 for WB, IP and IF) and HA‐tag (H6908 for WB, IP and IF) were from Sigma (St. Louis, MO, USA); HRP‐conjugated anti‐Rabbit IgG (#111‐035‐003) and HRP‐conjugated anti‐Mouse IgG (#115‐035‐003) secondary antibodies were used for WB analysis.

Techniques: Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Control, Expressing, Quantitative RT-PCR, Migration, Staining, MANN-WHITNEY

Journal: Clinical and Translational Medicine

Article Title: GRAMD4 inhibits tumour metastasis by recruiting the E3 ligase ITCH to target TAK1 for degradation in hepatocellular carcinoma

doi: 10.1002/ctm2.635

Figure Lengend Snippet: GRAMD4 facilitated the K48‐polyubiquitination‐dependent degradation of TAK1. (A) HEK293T cells were transfected with TAK1 (1 μg) and various concentrations of GRAMD4‐Flag plasmids. After 48 h, cell lysates were subjected to immunoblotting with anti‐Flag, anti‐HA and anti‐GAPDH antibodies. (B) HLF cells were transfected with various concentrations of GRAMD4‐Flag plasmid. After 48 h, cell lysates were subjected to immunoblotting with anti‐Flag, anti‐TAK1 and anti‐GAPDH antibodies. (C) HLF cells were transfected with GRAMD4 or vector control and were cultured for 24 h before being further incubated with CHX (20 μg/mL) for 0, 3, 6, 9 and 12 h. The TAK1 protein levels of the transfected cells were determined by western blot analysis. (D) HLF‐GRAMD4 or control cells were treated with MG132 (10 μM) for 6 h before immunoblot analysis for TAK1 and GRAMD4 levels. (E) HLF‐GRAMD4 or control cells were treated with Mg132 (10 μM) for 6 h, then TAK1 ubiquitination was measured via immunoprecipitation (IP) of TAK1. (F) Co‐IP analysis of ubiquitination of TAK1 in HEK293T cells co‐transfected with GRAMD4‐Myc plasmid, TAK1‐Flag plasmid and HA‐Ub‐K48, HA‐Ub‐WT, or HA‐Ub‐K63 plasmid

Article Snippet: Primary antibodies against GRAMD4 (24299‐1‐AP for WB, immunohistochemistry (IHC), immunofluorescence (IF) and (IP) and ITCH (20920‐1‐AP for WB, IP) were purchased from Proteintech (Wuhan, Hubei, China); primary antibodies against TAK1 (#5206 for WB, IHC, IF and IP), ERK (#4695 for WB), phospho‐ERK (#4370 for WB), p38 ((#8690 for WB), phospho‐p38 (#9215 for WB), JNK (#9252 for WB), phospho‐JNK(#9255 for WB) and Myc‐tag (#2276 for WB) were from CST (Danvers, MA, USA); Mouse serum IgG (I5381 for IP control), Rabbit serum IgG (I5006 for IP control), primary antibodies against Flag‐tag (F1804 for WB, IP and IF) and HA‐tag (H6908 for WB, IP and IF) were from Sigma (St. Louis, MO, USA); HRP‐conjugated anti‐Rabbit IgG (#111‐035‐003) and HRP‐conjugated anti‐Mouse IgG (#115‐035‐003) secondary antibodies were used for WB analysis.

Techniques: Transfection, Western Blot, Plasmid Preparation, Control, Cell Culture, Incubation, Ubiquitin Proteomics, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Clinical and Translational Medicine

Article Title: GRAMD4 inhibits tumour metastasis by recruiting the E3 ligase ITCH to target TAK1 for degradation in hepatocellular carcinoma

doi: 10.1002/ctm2.635

Figure Lengend Snippet: GRAMD4 recruited the E3 ligase ITCH to target TAK1 for K48‐linked ubiquitination and degradation. (A) 293T cells were transfected with vector or GRAMD4‐Flag plasmid together with control‐siRNA or ITCH‐siRNAs and analysed for TAK1, ITCH and Flag‐tag expression by western blot. (B) HLF cells with vector and GRAMD4 overexpression were transiently transfected with control‐siRNA or ITCH‐siRNAs and analysed for TAK1, ITCH and GRAMD4 expression by western blot. (C) Co‐IP analysis of the binding between exogenous GRAMD4‐Flag and ITCH‐HA in co‐transfected HEK293T cells. (D) Immunoassay of lysates from HLF cells, followed by immunoprecipitation with an anti‐TAK1 antibody. Co‐IP assay of binding between TAK1 and ITCH was performed at exogenous (E) endogenous (F) levels when GRAMD4 was overexpressed or not. Immunoassay of 293T cells transfected with expression vectors for various combinations of AMD4‐Myc, TAK1‐Flag, HA‐Ub (WT)(G), Ub (K48)(H) and Ub (K63)(I) together with control‐siRNA or ITCH‐siRNA, followed by immunoprecipitation of lysates with an anti‐Flag antibody and immunoblot analysis with anti‐Myc, anti‐HA, anti‐Flag anti‐ITCH antibodies

Article Snippet: Primary antibodies against GRAMD4 (24299‐1‐AP for WB, immunohistochemistry (IHC), immunofluorescence (IF) and (IP) and ITCH (20920‐1‐AP for WB, IP) were purchased from Proteintech (Wuhan, Hubei, China); primary antibodies against TAK1 (#5206 for WB, IHC, IF and IP), ERK (#4695 for WB), phospho‐ERK (#4370 for WB), p38 ((#8690 for WB), phospho‐p38 (#9215 for WB), JNK (#9252 for WB), phospho‐JNK(#9255 for WB) and Myc‐tag (#2276 for WB) were from CST (Danvers, MA, USA); Mouse serum IgG (I5381 for IP control), Rabbit serum IgG (I5006 for IP control), primary antibodies against Flag‐tag (F1804 for WB, IP and IF) and HA‐tag (H6908 for WB, IP and IF) were from Sigma (St. Louis, MO, USA); HRP‐conjugated anti‐Rabbit IgG (#111‐035‐003) and HRP‐conjugated anti‐Mouse IgG (#115‐035‐003) secondary antibodies were used for WB analysis.

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Control, FLAG-tag, Expressing, Western Blot, Over Expression, Co-Immunoprecipitation Assay, Binding Assay, Immunoprecipitation

Journal: Clinical and Translational Medicine

Article Title: GRAMD4 inhibits tumour metastasis by recruiting the E3 ligase ITCH to target TAK1 for degradation in hepatocellular carcinoma

doi: 10.1002/ctm2.635

Figure Lengend Snippet: GRAMD4 and TAK1 had an inverse correlation in HCC tissues. (A) Western blot analysis of GRAMD4 and TAK1 expression in HCC and non‐cancerous tissues, GAPDH was used as a loading control. (B) Correlation analysis of GRAMD4 and TAK1 expression according to western blot analysis in HCC tissues from the investigated HCC patients. (C) IHC staining of GRAMD4 and TAK1 expression in HCC tissues from clinical HCC samples. The images are representative of figures from the investigated HCC patients (Scale bar: 250 μm, 25 μm). (D) Correlation analysis of GRAMD4 and TAK1 expression according to IHC staining in HCC tissues from the investigated HCC patients. (E‐F) Kaplan–Meier analysis of the overall and disease‐free survival of HCC patients stratified by GRAMD4/TAK1 expression levels. Statistical analysis was performed using Pearson's correlation in (B, D). (G) A proposed model for the mechanism through which GRAMD4 downregulates TAK1 by recruiting ITCH, resulting in the deactivation of the MAPK signalling pathway and inhibition of HCC progression and metastasis

Article Snippet: Primary antibodies against GRAMD4 (24299‐1‐AP for WB, immunohistochemistry (IHC), immunofluorescence (IF) and (IP) and ITCH (20920‐1‐AP for WB, IP) were purchased from Proteintech (Wuhan, Hubei, China); primary antibodies against TAK1 (#5206 for WB, IHC, IF and IP), ERK (#4695 for WB), phospho‐ERK (#4370 for WB), p38 ((#8690 for WB), phospho‐p38 (#9215 for WB), JNK (#9252 for WB), phospho‐JNK(#9255 for WB) and Myc‐tag (#2276 for WB) were from CST (Danvers, MA, USA); Mouse serum IgG (I5381 for IP control), Rabbit serum IgG (I5006 for IP control), primary antibodies against Flag‐tag (F1804 for WB, IP and IF) and HA‐tag (H6908 for WB, IP and IF) were from Sigma (St. Louis, MO, USA); HRP‐conjugated anti‐Rabbit IgG (#111‐035‐003) and HRP‐conjugated anti‐Mouse IgG (#115‐035‐003) secondary antibodies were used for WB analysis.

Techniques: Western Blot, Expressing, Control, Immunohistochemistry, Inhibition