human keap1 cdna  (Addgene inc)

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15 associates with Keap1. A Network graph representation of interaction from the BioGRID for TRIM15. Users can select the ‘Network’ tab from the ‘Switch View’ menu to view interactions data when available. B Immunoblot detection of the indicated proteins in a Co-IP assay performed in HEK293 cells. C Immunofluorescence colocalization of TRIM15 with Keap1 in H1299 and H1975 was assessed by rabbit anti-TRIM15 detected with anti-rabbit IgG-Alexa Fluor 488 (green fluorescence), and detection of Keap1 with mouse anti-Keap1 detected with anti-mouse IgG Alexa 594 (red fluorescence). The colocalization of TRIM15 and Keap1 is illustrated by overlay of the images, illustrated by yellow fluorescence. D Immunoprecipitation assay revealing the interaction between TRIM15 with Keap1 in H1299 and H1975 cells. E Co-localization of TRIM15 (green) and Keap1 (red) in 2 NSCLC tissues from two patients by immunofluorescent confocal microscopy

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Immunofluorescence, Fluorescence, Immunoprecipitation, Confocal Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15 promotes Keap1 ubiquitination and degradation. A Immunoblot analysis of Keap1, Flag-TRIM15, and β-actin in HEK293 cells transfected with expression vector for Flag-TRIM15 or with empty vector, with or without treatment of 10 μM MG132. Mutations of Flag-TRIM15-ΔRING impaired the ability of TRIM15 to degrade Keap1 protein in HEK293 cells. B HEK293 cells were transfected with the indicated plasmids. The cells were treated with 50 μg/ml CHX for indicated time periods. Densitometry analysis performed on corresponding immunoblots to assess Keap1 half-life in the indicated conditions. C H1299 or H1650 cells were transfected with the indicated plasmids, treated with 50 μg/ml CHX, harvested at different time points, and then immunoblotted with using the indicated antibodies. Densitometry analysis performed on corresponding immunoblots to assess Keap1 half-life in the indicated conditions. D HEK293 cells were transfected with Flag-TRIM15, Flag-TRIM15-ΔRING, HA-Keap1, and His-ubiquitin plasmids, and the cell lysates were subjected to immunoprecipitation using anti-HA antibodies (left panel) or Ni–NTA pull-down (right panel) under denaturing conditions, followed by immunoblotting with the indicated antibodies. Overexpression of wild-type but not the mutant TRIM15-ΔRING promoted ubiquitination of Keap1. E Knockdown of endogenous TRIM15 decreased the ubiquitination of HA-Keap1 in HEK293 cells analyzed by in vitro ubiquitination assays. F Cell lysates prepared in D were immunoprecipitated with anti-HA. knockdown of TRIM15 inhibits K48-linked but not K63-linked ubiquitination of HA-Keap1

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Over Expression, Mutagenesis, Knockdown, In Vitro

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15 stabilizes Nrf2 through binding with Keap1. A HEK293 cells transfected with Flag-TRIM15, HA-Keap1, and Myc-Nrf2 were subjected to immunoprecipitation with HA antibody. Lysates were analyzed by western blotting. B TRIM15 reduced the interaction between Nrf2 and Keap1. Cell lysates were immunoprecipitated with an anti-Keap1 antibody and blotted with an anti-Nrf2 antibody. C – F Subcellular fractionation was used to isolate cytoplasmic and nuclear proteins, and immunoblotting was performed to examine the localization of Nrf2 following the downregulation or overexpression of TRIM15. Nuclear and cytoplasmic levels of Nrf2 are quantified. G Effect of TRIM15 knockdown (H1299 cells) or overexpression (H1650 cells) on the mRNA expression of the Nrf2-regulated genes. NAD(P)H quinone dehydrogenase1(NOQ1), thioredoxin (TXN), peroxiredoxin 1(PRDX1), hemoxygenase 1(HMOX1), glutamate-cysteine ligase catalytic subunit (GCLC), glutathione S-transferase μ1(GSTM1), glutathione S-transferase μ3(GSTM3), ferritin light chain (FTL). H Representative IHC staining images of Nrf2 in the same set of NSCLC tissue slices. Correlation analysis of TRIM15 and Nrf2 expression in NSCLC samples. Spearman correlation coefficients are shown. Scale bars, 100 μm. Statistical analyses were performed by two-tailed unpaired Student’s t -test. ** P < 0.01

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot, Fractionation, Over Expression, Knockdown, Expressing, Immunohistochemistry, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15-mediated Nrf2 signaling regulates growth and invasion in NSCLC cells in vitro. A Western blot analyses of TRIM15, Nrf2, Keap1, and Nrf2 target NQO1 in H1299 cells with TRIM15 knockdown with or without subsequent Nrf2 overexpression and H1650 cells overexpressing TRIM15 with or without subsequent knockdown of Nrf2. B ARE Luc reporter activity assessed in H1299 cells expressing shTRIM15, sh TRIM15 + Nrf2 or H1650 cells overexpressing TRIM15 with or without subsequent knockdown of Nrf2. Up-regulating of Nrf2 expression in H1299 cells or down-regulating of Nrf2 expression in H1650 cells was set as a control. C – F Cell proliferation ( C , D ), invasion ( E ) and ROS formation ( F ) in H1299 cells with or without shTRIM15 and Nrf2 rescue or in H1650 cells with or without TRIM15 overexpression and shNrf2 rescue. Statistical analyses were performed by two-tailed unpaired Student’s t -test. ** P < 0.01

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: In Vitro, Western Blot, Knockdown, Over Expression, Activity Assay, Expressing, Control, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15 mediated increase in Nrf2 regulates growth and invasion in vivo. A Nude mice were randomized into three groups and subcutaneously injected with H1650 cells that had been transfected with control (empty vector), TRIM15, or TRIM15 + shNrf2 plasmids. Tumors formed in nude mice were collected 30 days after grafting, and the tumor weight were measured. B Measurement of tumor volume in experimental groups over time. C Western blotting analysis was performed to evaluate the levels of TRIM15, Nrf2, Keap1, and NQO1 in harvested tumors. D , E Up-regulation of TRIM15 significantly promoted lung metastasis in H1650 xenograft nude mice models, whereas the suppression of Nrf2 prevented the tumor metastasis of TRIM15 overexpressing cells. Representative pictures of the lung metastases in nude mice by H&E staining. Quantification of lung metastases in all groups. Scale bar: 200 μm. F , G A representative image of tumor growth in nude mice subcutaneously inoculated with H1299 cells tranfected with shCtrl, shTRIM15 or shTRIM15 + Nrf2 plasmids. Tumor volumes were measured on the indicated days. H Western blotting analysis was performed to evaluate the levels of TRIM15, Nrf2, Keap1, and NQO1 in xenograft tumors. I Representative pictures of the lung metastases in nude mice by H&E staining. Quantification of lung metastases in all groups. Scale bar: 200 μm. J TRIM15 was significantly upregulated in NSCLC and that increased TRIM15 was associated with poor survival. TRIM15 promoted tumor proliferation and metastasis by activating Nrf2 signaling. Furthermore, TRIM15 regulated Nrf2 activity by modulating Keap1 and inducing its ubiquitination and degradation in NSCLC cells. Activation of Nrf2 facilitated tumor cell proliferation and invasion. N.S. represents no significant. Statistical analyses were performed by two-tailed unpaired Student’s t -test. ** P < 0.01

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: In Vivo, Injection, Transfection, Control, Plasmid Preparation, Western Blot, Staining, Activity Assay, Activation Assay, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15 is upregulated in NSCLC and associated with NSCLC progression. A The gene expression of TRIM15 between NSCLC samples and normal lung tissue from GEPIA. LUAD (Lung adenocarcinoma), LUSC (Lung squamous cell carcinoma). B Western blot analysis of TRIM15 expression in NSCLC tissues compared with corresponding noncancerous lung tissues. C IHC analysis of TRIM15 expression in 2 NSCLC samples. Representative images are shown. Scale bar, 100 μm. D Representative IHC images of TRIM15 in NSCLC tissues with or without distant metastasis. Scale bar, 200 μm. E OS analysis of patients with NSCLC stratified by the TRIM15 expression level in 98 samples. Patients with high-expression levels of TRIM15 had shorter OS times than patients with low expression levels. ** P < 0.01

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: Expressing, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: Roles of TRIM15 in promoting NSCLC growth and metastasis. A Expression levels of TRIM15 in the indicated lung cancer cell lines were analysed by western blotting. Confirmation of TRIM15 knockdown in H1299. H1650 cells were transfected with control plasmids or vector expressing TRIM15 or TRIM15-ΔRING. Immunoblot detection of TRIM15 and TRIM15-ΔRING expression in H1650 cells. B , C The effect of TRIM15 knockdown and overexpression on NSCLC cells proliferation was assessed by CCK8 and EdU-incorporation assay. D , E The effects of TRIM15 loss- or gain-of-function on migration ( D ) and invasion ( E ) of NSCLC cells. Knockdown of TRIM15 resulted in significant inhibited migration ( D ) and invasion ( E ) of H1299 cells. TRIM15 upregulation significantly increased cancer cell migration ( D ) and invasion ( E ), while the TRIM15-ΔRING lost this ability. Statistical analyses were performed by two-tailed unpaired Student’s t -test. ** P < 0.01

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: Expressing, Western Blot, Knockdown, Transfection, Control, Plasmid Preparation, Over Expression, Migration, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15 associates with Keap1. A Network graph representation of interaction from the BioGRID for TRIM15. Users can select the ‘Network’ tab from the ‘Switch View’ menu to view interactions data when available. B Immunoblot detection of the indicated proteins in a Co-IP assay performed in HEK293 cells. C Immunofluorescence colocalization of TRIM15 with Keap1 in H1299 and H1975 was assessed by rabbit anti-TRIM15 detected with anti-rabbit IgG-Alexa Fluor 488 (green fluorescence), and detection of Keap1 with mouse anti-Keap1 detected with anti-mouse IgG Alexa 594 (red fluorescence). The colocalization of TRIM15 and Keap1 is illustrated by overlay of the images, illustrated by yellow fluorescence. D Immunoprecipitation assay revealing the interaction between TRIM15 with Keap1 in H1299 and H1975 cells. E Co-localization of TRIM15 (green) and Keap1 (red) in 2 NSCLC tissues from two patients by immunofluorescent confocal microscopy

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Immunofluorescence, Fluorescence, Immunoprecipitation, Confocal Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15 promotes Keap1 ubiquitination and degradation. A Immunoblot analysis of Keap1, Flag-TRIM15, and β-actin in HEK293 cells transfected with expression vector for Flag-TRIM15 or with empty vector, with or without treatment of 10 μM MG132. Mutations of Flag-TRIM15-ΔRING impaired the ability of TRIM15 to degrade Keap1 protein in HEK293 cells. B HEK293 cells were transfected with the indicated plasmids. The cells were treated with 50 μg/ml CHX for indicated time periods. Densitometry analysis performed on corresponding immunoblots to assess Keap1 half-life in the indicated conditions. C H1299 or H1650 cells were transfected with the indicated plasmids, treated with 50 μg/ml CHX, harvested at different time points, and then immunoblotted with using the indicated antibodies. Densitometry analysis performed on corresponding immunoblots to assess Keap1 half-life in the indicated conditions. D HEK293 cells were transfected with Flag-TRIM15, Flag-TRIM15-ΔRING, HA-Keap1, and His-ubiquitin plasmids, and the cell lysates were subjected to immunoprecipitation using anti-HA antibodies (left panel) or Ni–NTA pull-down (right panel) under denaturing conditions, followed by immunoblotting with the indicated antibodies. Overexpression of wild-type but not the mutant TRIM15-ΔRING promoted ubiquitination of Keap1. E Knockdown of endogenous TRIM15 decreased the ubiquitination of HA-Keap1 in HEK293 cells analyzed by in vitro ubiquitination assays. F Cell lysates prepared in D were immunoprecipitated with anti-HA. knockdown of TRIM15 inhibits K48-linked but not K63-linked ubiquitination of HA-Keap1

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Over Expression, Mutagenesis, Knockdown, In Vitro

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15 stabilizes Nrf2 through binding with Keap1. A HEK293 cells transfected with Flag-TRIM15, HA-Keap1, and Myc-Nrf2 were subjected to immunoprecipitation with HA antibody. Lysates were analyzed by western blotting. B TRIM15 reduced the interaction between Nrf2 and Keap1. Cell lysates were immunoprecipitated with an anti-Keap1 antibody and blotted with an anti-Nrf2 antibody. C – F Subcellular fractionation was used to isolate cytoplasmic and nuclear proteins, and immunoblotting was performed to examine the localization of Nrf2 following the downregulation or overexpression of TRIM15. Nuclear and cytoplasmic levels of Nrf2 are quantified. G Effect of TRIM15 knockdown (H1299 cells) or overexpression (H1650 cells) on the mRNA expression of the Nrf2-regulated genes. NAD(P)H quinone dehydrogenase1(NOQ1), thioredoxin (TXN), peroxiredoxin 1(PRDX1), hemoxygenase 1(HMOX1), glutamate-cysteine ligase catalytic subunit (GCLC), glutathione S-transferase μ1(GSTM1), glutathione S-transferase μ3(GSTM3), ferritin light chain (FTL). H Representative IHC staining images of Nrf2 in the same set of NSCLC tissue slices. Correlation analysis of TRIM15 and Nrf2 expression in NSCLC samples. Spearman correlation coefficients are shown. Scale bars, 100 μm. Statistical analyses were performed by two-tailed unpaired Student’s t -test. ** P < 0.01

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot, Fractionation, Over Expression, Knockdown, Expressing, Immunohistochemistry, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15-mediated Nrf2 signaling regulates growth and invasion in NSCLC cells in vitro. A Western blot analyses of TRIM15, Nrf2, Keap1, and Nrf2 target NQO1 in H1299 cells with TRIM15 knockdown with or without subsequent Nrf2 overexpression and H1650 cells overexpressing TRIM15 with or without subsequent knockdown of Nrf2. B ARE Luc reporter activity assessed in H1299 cells expressing shTRIM15, sh TRIM15 + Nrf2 or H1650 cells overexpressing TRIM15 with or without subsequent knockdown of Nrf2. Up-regulating of Nrf2 expression in H1299 cells or down-regulating of Nrf2 expression in H1650 cells was set as a control. C – F Cell proliferation ( C , D ), invasion ( E ) and ROS formation ( F ) in H1299 cells with or without shTRIM15 and Nrf2 rescue or in H1650 cells with or without TRIM15 overexpression and shNrf2 rescue. Statistical analyses were performed by two-tailed unpaired Student’s t -test. ** P < 0.01

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: In Vitro, Western Blot, Knockdown, Over Expression, Activity Assay, Expressing, Control, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15 mediated increase in Nrf2 regulates growth and invasion in vivo. A Nude mice were randomized into three groups and subcutaneously injected with H1650 cells that had been transfected with control (empty vector), TRIM15, or TRIM15 + shNrf2 plasmids. Tumors formed in nude mice were collected 30 days after grafting, and the tumor weight were measured. B Measurement of tumor volume in experimental groups over time. C Western blotting analysis was performed to evaluate the levels of TRIM15, Nrf2, Keap1, and NQO1 in harvested tumors. D , E Up-regulation of TRIM15 significantly promoted lung metastasis in H1650 xenograft nude mice models, whereas the suppression of Nrf2 prevented the tumor metastasis of TRIM15 overexpressing cells. Representative pictures of the lung metastases in nude mice by H&E staining. Quantification of lung metastases in all groups. Scale bar: 200 μm. F , G A representative image of tumor growth in nude mice subcutaneously inoculated with H1299 cells tranfected with shCtrl, shTRIM15 or shTRIM15 + Nrf2 plasmids. Tumor volumes were measured on the indicated days. H Western blotting analysis was performed to evaluate the levels of TRIM15, Nrf2, Keap1, and NQO1 in xenograft tumors. I Representative pictures of the lung metastases in nude mice by H&E staining. Quantification of lung metastases in all groups. Scale bar: 200 μm. J TRIM15 was significantly upregulated in NSCLC and that increased TRIM15 was associated with poor survival. TRIM15 promoted tumor proliferation and metastasis by activating Nrf2 signaling. Furthermore, TRIM15 regulated Nrf2 activity by modulating Keap1 and inducing its ubiquitination and degradation in NSCLC cells. Activation of Nrf2 facilitated tumor cell proliferation and invasion. N.S. represents no significant. Statistical analyses were performed by two-tailed unpaired Student’s t -test. ** P < 0.01

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: In Vivo, Injection, Transfection, Control, Plasmid Preparation, Western Blot, Staining, Activity Assay, Activation Assay, Two Tailed Test

Journal: Redox Biology

Article Title: Chemoproteomic profiling reveals cellular targets of nitro-fatty acids

doi: 10.1016/j.redox.2021.102126

Figure Lengend Snippet: Fluorescent detection of nitro-alkylated proteins in living cells. (A) Workflow for in-gel fluorescence detection of nitro-alkylated proteins using the alk-9-NO 2 -OA probe 8 and azide-rhodamine. CuAAC, copper-catalyzed azide-alkyne cycloaddition. (B) Time-dependent labelling of endogenous proteins and overexpressed HA-KEAP1 in HEK293FT cells at 2.5 μM alk-9-NO 2 -OA. (C) Dose-dependent labelling of endogenous proteins and overexpressed HA-KEAP1 in HEK293FT cells after treatment with indicated concentrations of alk-9-NO 2 -OA for 1 h. (D) Labeling of wildtype (wt) KEAP1 and its mutants in HEK293T cells at 2.5 μM alk-9-NO 2 -OA for 1 h. 3M and 7M refer to mutants with 3 and 7 cysteines mutated, respectively. (E) Fluorescent detection of nitro-alkylated proteins in different human cell lines treated with 10 μM alk-9-NO 2 -OA for 15 min. Anti-HA, anti-GAPDH, anti-tubulin blots and Coomassie stains act as loading controls for the accompanying fluorescence gels. Selected protein molecular weight markers are indicated for each gel.

Article Snippet: The following mutations to pCMV3-HA-KEAP1 (Sino Biological, HG11981-NY) were made using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Stratagene) and validated by Sanger sequencing ( ).

Techniques: Fluorescence, Labeling, Molecular Weight