peptide synthesizer 433 Search Results


nb100  (Novus Biologicals)
94
Novus Biologicals nb100
Nb100, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/peptide+synthesizer+433/HDAC1+Antibody/pm34383982-118-61-63
Average 94 stars, based on 1 article reviews
nb100 - by Bioz Stars, 2026-09
94/100 stars
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anti hdac1  (Bio-Techne corporation)
94
Bio-Techne corporation anti hdac1
Anti Hdac1, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/peptide+synthesizer+433/HDAC1+Antibody/pmc08699676-100-31-33
Average 94 stars, based on 1 article reviews
anti hdac1 - by Bioz Stars, 2026-09
94/100 stars
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primary antibodies against g55  (Novus Biologicals)
92
Novus Biologicals primary antibodies against g55
(A) Volcano plot of mRNAs (dots). Differences between LUAD (n = 515) and normal lung (n = 51) in TCGA expressed as a P value (y axis) and a t-statistic (x axis) to identify genes upregulated (t > 0) or downregulated (t < 0) in cancer. <t>G55</t> indicated. (B) Quantification of G55 mRNA in tumors (red dots) and normal tissues (green dots). Tumor types significantly different from normal (asterisks). Mean values (horizontal bars). (C) G55 mRNA levels in the TCGA pan-cancer cohort (dots) annotated on the basis of mutations. “Unaligned” indicates no correlation with mutations. Sample sizes indicated (n). Tumor types color coded. (D) Kaplan-Meier analysis of TCGA pan-cancer cohort (n = 9,502) scored based on G55 mRNA expression. High: top third. Other: middle and bottom thirds. (E) Western blot (WB) analysis of G55 in parental (WT) and TP53-KO A549 clones. Cells treated with vehicle (DMSO) or nutlin-3 to enhance p53 levels. α-Tubulin loading control. (F) Quantitative real-time PCR (qPCR) analysis of miR-transfected cells. Negative control (miR-NC). (G) WB analysis of miR-transfected cells. β-Actin loading control. (H and I) qPCR (H) and WB (I) analysis of A549 cells cotransfected with shRNAs and miRs. Control vectors indicated by (–). Values relative to control transfectants. (J and K) qPCR (J) and WB (K) analysis of H1299 cells cotransfected with p53 expression vector and antagomir-34a (anti-34a). Vec, empty vector; anti-NC, control antagomir. (L) Reporter assays on cells cotransfected with miRs and reporters containing G55 3′-UTRs with intact (WT) or mutant (MT) miR-34a binding sites. Values relative to miR-NC. n = 4. (M) RNA polymerase II ChIP assays on GORASP2 gene promoter. Values are percentage of input (total chromatin). All WB densitometric values are relative to control. Results represent mean ± SD. n = 3, unless otherwise indicated. P values: ANOVA (H and J), univariate Cox (D), 2-sided t test (all others).
Primary Antibodies Against G55, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/peptide+synthesizer+433/GRASP55+Antibody/pmc07773359-449-2-8
Average 92 stars, based on 1 article reviews
primary antibodies against g55 - by Bioz Stars, 2026-09
92/100 stars
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total fak  (Cell Signaling Technology Inc)
95
Cell Signaling Technology Inc total fak
PLCD1 ectopic expression inhibited oncogenic signaling. a Experimentally expressed PLCD1 in 293 T cells was confirmed by Western blot. b Activity of several oncogenic signaling reporters were evaluated using dual-luciferase reporters assay in 293 T cells. b TCF activity and CCND1 , c-Myc <t>and</t> <t>MMP7</t> promoter reporter activities in PLCD1 -overexpressing A498 (upper) and HH244 (bottom) cells. d Western blot analyses of total β-catenin, p-β-catenin (Ser552), c-Myc, active β-catenin, cyclinD1, MMP7, PLCD1 and GAPDH. e Western blot analyses of EGFR, ERK1/2, Src, <t>FAK</t> and PLCD1 , with GAPDH as internal control. Histograms indicated quantification of the phosphorylated protein normalized to corresponding total protein level. f Schematic diagram of roles and mechanisms of PLCD1 in RCC tumorigenesis
Total Fak, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/peptide+synthesizer+433/FAK+XP+Rabbit+mAb/pmc09972803-113-38-40
Average 95 stars, based on 1 article reviews
total fak - by Bioz Stars, 2026-09
95/100 stars
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total tbc1d1  (Cell Signaling Technology Inc)
93
Cell Signaling Technology Inc total tbc1d1
DKI mice have reduced skeletal muscle AMPK α and β2 content but intact AMPK, ACC and <t>TBC1D1</t> signaling in response to a maximal running test at a 5° incline versus WT. Skeletal muscles were collected from rested and maximally exercised male WT and DKI mice, and phosphorylation of AMPK and downstream substrates was assessed using Western blotting. (A) Representative immunoblots of p-T172 and total AMPK, p-S182 and total AMPK β, p-S79 and total acetyl-CoA carboxylase (ACC) and p-S660 and total TBC1 domain family member 1 (TBC1D1) with representative stain free image. Quantified relative (B) AMPK p-T172, (C) AMPK β p-S182, (D) ACC p-S79, and (E) TBC1D1 p-S660; (F) Total AMPK α; (G) Total AMPK β. Male mice, 17–20 weeks, n = 6–8. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Total Tbc1d1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/peptide+synthesizer+433/TBC1D1+Rabbit+mAb/pmc08980720-163-25-45
Average 93 stars, based on 1 article reviews
total tbc1d1 - by Bioz Stars, 2026-09
93/100 stars
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free peptide (amino acids 433 to 450 of mouse slc52a2)  (GenScript corporation)
90
GenScript corporation free peptide (amino acids 433 to 450 of mouse slc52a2)
DKI mice have reduced skeletal muscle AMPK α and β2 content but intact AMPK, ACC and <t>TBC1D1</t> signaling in response to a maximal running test at a 5° incline versus WT. Skeletal muscles were collected from rested and maximally exercised male WT and DKI mice, and phosphorylation of AMPK and downstream substrates was assessed using Western blotting. (A) Representative immunoblots of p-T172 and total AMPK, p-S182 and total AMPK β, p-S79 and total acetyl-CoA carboxylase (ACC) and p-S660 and total TBC1 domain family member 1 (TBC1D1) with representative stain free image. Quantified relative (B) AMPK p-T172, (C) AMPK β p-S182, (D) ACC p-S79, and (E) TBC1D1 p-S660; (F) Total AMPK α; (G) Total AMPK β. Male mice, 17–20 weeks, n = 6–8. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Free Peptide (Amino Acids 433 To 450 Of Mouse Slc52a2), supplied by GenScript corporation, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/peptide+synthesizer+433/free+peptide++amino+acids+433+to+450+of+mouse+slc52a2+/pm26791833-91-10-24
Average 90 stars, based on 1 article reviews
free peptide (amino acids 433 to 450 of mouse slc52a2) - by Bioz Stars, 2026-09
90/100 stars
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cd79a antibody jcb117 hm47 a9  (Novus Biologicals)
N/A
The CD79A Antibody JCB117 HM47 A9 from Novus Biologicals is a mouse monoclonal antibody to CD79A This antibody reacts with human mouse rat porcine bovine primate The CD79A Antibody JCB117 HM47 A9 has been validated
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zbtb12 antibody  (Novus Biologicals)
N/A
The ZBTB12 Antibody from Novus Biologicals is a rabbit polyclonal antibody to ZBTB12 This antibody reacts with human The ZBTB12 Antibody has been validated for the following applications Western Blot
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raldh2 antibody (biotin)  (biorbyt)
N/A
Rabbit polyclonal antibody against RALDH2 conjugated to Biotin Isotype Note: IgG Host Note: Rabbit Conjugation Note: Biotin Reactivity Note: Human, Mouse, Rat
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recombinant g3bp antibody  (Aladdin Scientific Corporation)
N/A
Rabbit anti Human G3BP Antibody, Recombinant, could be used for WB, IF/ICC and IHC.Application:WB: 1/500 – 1/1000IF/ICC: 1/50 – 1/200IHC: 1/50 – 1/100Protein FunctionMay be a regulated effector of stress granule assembly. Phosphorylation-dependent sequence-specific endoribonuclease
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cp antibody - n-terminal region (oaab04336)  (Aviva Systems)
N/A
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loc443300 antibody - n-terminal region (oasa06932)  (Aviva Systems)
N/A
RABBIT ANTI 14-3-3 EPSILON (N-TERMINAL); RABBIT ANTI 14-3-3 EPSILON (N-TERMINAL)_x000D_
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Image Search Results


(A) Volcano plot of mRNAs (dots). Differences between LUAD (n = 515) and normal lung (n = 51) in TCGA expressed as a P value (y axis) and a t-statistic (x axis) to identify genes upregulated (t > 0) or downregulated (t < 0) in cancer. G55 indicated. (B) Quantification of G55 mRNA in tumors (red dots) and normal tissues (green dots). Tumor types significantly different from normal (asterisks). Mean values (horizontal bars). (C) G55 mRNA levels in the TCGA pan-cancer cohort (dots) annotated on the basis of mutations. “Unaligned” indicates no correlation with mutations. Sample sizes indicated (n). Tumor types color coded. (D) Kaplan-Meier analysis of TCGA pan-cancer cohort (n = 9,502) scored based on G55 mRNA expression. High: top third. Other: middle and bottom thirds. (E) Western blot (WB) analysis of G55 in parental (WT) and TP53-KO A549 clones. Cells treated with vehicle (DMSO) or nutlin-3 to enhance p53 levels. α-Tubulin loading control. (F) Quantitative real-time PCR (qPCR) analysis of miR-transfected cells. Negative control (miR-NC). (G) WB analysis of miR-transfected cells. β-Actin loading control. (H and I) qPCR (H) and WB (I) analysis of A549 cells cotransfected with shRNAs and miRs. Control vectors indicated by (–). Values relative to control transfectants. (J and K) qPCR (J) and WB (K) analysis of H1299 cells cotransfected with p53 expression vector and antagomir-34a (anti-34a). Vec, empty vector; anti-NC, control antagomir. (L) Reporter assays on cells cotransfected with miRs and reporters containing G55 3′-UTRs with intact (WT) or mutant (MT) miR-34a binding sites. Values relative to miR-NC. n = 4. (M) RNA polymerase II ChIP assays on GORASP2 gene promoter. Values are percentage of input (total chromatin). All WB densitometric values are relative to control. Results represent mean ± SD. n = 3, unless otherwise indicated. P values: ANOVA (H and J), univariate Cox (D), 2-sided t test (all others).

Journal: The Journal of Clinical Investigation

Article Title: A protumorigenic secretory pathway activated by p53 deficiency in lung adenocarcinoma

doi: 10.1172/JCI137186

Figure Lengend Snippet: (A) Volcano plot of mRNAs (dots). Differences between LUAD (n = 515) and normal lung (n = 51) in TCGA expressed as a P value (y axis) and a t-statistic (x axis) to identify genes upregulated (t > 0) or downregulated (t < 0) in cancer. G55 indicated. (B) Quantification of G55 mRNA in tumors (red dots) and normal tissues (green dots). Tumor types significantly different from normal (asterisks). Mean values (horizontal bars). (C) G55 mRNA levels in the TCGA pan-cancer cohort (dots) annotated on the basis of mutations. “Unaligned” indicates no correlation with mutations. Sample sizes indicated (n). Tumor types color coded. (D) Kaplan-Meier analysis of TCGA pan-cancer cohort (n = 9,502) scored based on G55 mRNA expression. High: top third. Other: middle and bottom thirds. (E) Western blot (WB) analysis of G55 in parental (WT) and TP53-KO A549 clones. Cells treated with vehicle (DMSO) or nutlin-3 to enhance p53 levels. α-Tubulin loading control. (F) Quantitative real-time PCR (qPCR) analysis of miR-transfected cells. Negative control (miR-NC). (G) WB analysis of miR-transfected cells. β-Actin loading control. (H and I) qPCR (H) and WB (I) analysis of A549 cells cotransfected with shRNAs and miRs. Control vectors indicated by (–). Values relative to control transfectants. (J and K) qPCR (J) and WB (K) analysis of H1299 cells cotransfected with p53 expression vector and antagomir-34a (anti-34a). Vec, empty vector; anti-NC, control antagomir. (L) Reporter assays on cells cotransfected with miRs and reporters containing G55 3′-UTRs with intact (WT) or mutant (MT) miR-34a binding sites. Values relative to miR-NC. n = 4. (M) RNA polymerase II ChIP assays on GORASP2 gene promoter. Values are percentage of input (total chromatin). All WB densitometric values are relative to control. Results represent mean ± SD. n = 3, unless otherwise indicated. P values: ANOVA (H and J), univariate Cox (D), 2-sided t test (all others).

Article Snippet: We purchased primary antibodies against G55 (NB100-74431) from Novus Biologicals; against GM130 (560066) from BD Transduction Laboratories; against G45 (OAAN01805) from Aviva Systems Biology; against GOLPH3 (ab98023) from Abcam; against IGFBP2 (11065-3-AP), SPP1 (22952-1-AP), MYO18A (14611-1-AP), MAOA (10539-1-AP), and RAB6A (10187-2-AP) from Proteintech; against α-tubulin (T9026), EGFP (G6539) from MilliporeSigma; against PARP-1 (9542), hemagglutinin (3724 and 2367), β-actin (4970), p53 (9282), GM130 (12480), EEA1 (3288), ubiquitin (43124), and MYOIIA (3403) from Cell Signaling Technology; against ACBD3 (sc-101277), GRASP65 (sc-374423), and p53 (sc-126) from Santa Cruz Biotechnology; against calnexin (ab22595) and RAB11 (ab95375) from Abcam; and against RNA polymerase II (05-623, clone CTD4H8) from MilliporeSigma.

Techniques: Expressing, Western Blot, Clone Assay, Real-time Polymerase Chain Reaction, Transfection, Negative Control, Plasmid Preparation, Mutagenesis, Binding Assay

(A) WB analysis of TP53-mutant LUAD cell lines transfected with G55 (siG55#1, #2) or control (siCTL) siRNA. (B) Cell proliferation in monolayer culture quantified at 4 days by WST-1 assays. Results relative to siCTL controls. n = 5. (C) Anchorage-independent colony formation quantified after 10 days in soft agarose. Results relative to siCTL controls. (D) Boyden chamber assays. FBS (10%) added to bottom chamber as chemoattractant. Cells that migrated or invaded through porous filter (Matrigel-coated for invasion assays) photographed 8 hours after seeding in upper chamber and counted manually. Results relative to siCTL controls. (E) qPCR analysis of TP53-WT and TP53-mutant (MT) LUAD cell lines. Results relative to siCTL. (F and G) Anchorage-dependent colony formation. Cells seeded at low density on plastic. Results expressed as fractions (F) or percentage reductions (G) relative to siCTL in TP53-WT and TP53-mutant (MT) cell lines (G). (H) WB analysis of 344SQ cells transfected with G55 (shG55#1, #2, #3) or control (shCTL) shRNAs. G55 densitometric values relative to shCTL. (I) Primary subcutaneous tumor weight (left plot) and lung metastasis numbers (right plot) per mouse (dots). Necropsies performed 5 weeks after subcutaneous injection of 344SQ cells into syngeneic, immunocompetent mice. (J) Kaplan-Meier analysis of mouse cohorts bearing G55-deficient (shG55) or G55-replete (shCTL) 344SQ orthotopic lung tumors (n = 10 mice per cohort). (K) WB analysis of 344P LUAD cells transfected with GFP-tagged G55 or empty (Vec) expression vectors. (L) Primary tumor weights (left plot) and lung metastasis numbers (right plot) determined at necropsy 5 weeks after subcutaneous injection of tumor cells. Results represent mean ± SD. n = 3, unless otherwise indicated. P values: ANOVA (B–D and I), log-rank test (J), 2-sided t test (all others).

Journal: The Journal of Clinical Investigation

Article Title: A protumorigenic secretory pathway activated by p53 deficiency in lung adenocarcinoma

doi: 10.1172/JCI137186

Figure Lengend Snippet: (A) WB analysis of TP53-mutant LUAD cell lines transfected with G55 (siG55#1, #2) or control (siCTL) siRNA. (B) Cell proliferation in monolayer culture quantified at 4 days by WST-1 assays. Results relative to siCTL controls. n = 5. (C) Anchorage-independent colony formation quantified after 10 days in soft agarose. Results relative to siCTL controls. (D) Boyden chamber assays. FBS (10%) added to bottom chamber as chemoattractant. Cells that migrated or invaded through porous filter (Matrigel-coated for invasion assays) photographed 8 hours after seeding in upper chamber and counted manually. Results relative to siCTL controls. (E) qPCR analysis of TP53-WT and TP53-mutant (MT) LUAD cell lines. Results relative to siCTL. (F and G) Anchorage-dependent colony formation. Cells seeded at low density on plastic. Results expressed as fractions (F) or percentage reductions (G) relative to siCTL in TP53-WT and TP53-mutant (MT) cell lines (G). (H) WB analysis of 344SQ cells transfected with G55 (shG55#1, #2, #3) or control (shCTL) shRNAs. G55 densitometric values relative to shCTL. (I) Primary subcutaneous tumor weight (left plot) and lung metastasis numbers (right plot) per mouse (dots). Necropsies performed 5 weeks after subcutaneous injection of 344SQ cells into syngeneic, immunocompetent mice. (J) Kaplan-Meier analysis of mouse cohorts bearing G55-deficient (shG55) or G55-replete (shCTL) 344SQ orthotopic lung tumors (n = 10 mice per cohort). (K) WB analysis of 344P LUAD cells transfected with GFP-tagged G55 or empty (Vec) expression vectors. (L) Primary tumor weights (left plot) and lung metastasis numbers (right plot) determined at necropsy 5 weeks after subcutaneous injection of tumor cells. Results represent mean ± SD. n = 3, unless otherwise indicated. P values: ANOVA (B–D and I), log-rank test (J), 2-sided t test (all others).

Article Snippet: We purchased primary antibodies against G55 (NB100-74431) from Novus Biologicals; against GM130 (560066) from BD Transduction Laboratories; against G45 (OAAN01805) from Aviva Systems Biology; against GOLPH3 (ab98023) from Abcam; against IGFBP2 (11065-3-AP), SPP1 (22952-1-AP), MYO18A (14611-1-AP), MAOA (10539-1-AP), and RAB6A (10187-2-AP) from Proteintech; against α-tubulin (T9026), EGFP (G6539) from MilliporeSigma; against PARP-1 (9542), hemagglutinin (3724 and 2367), β-actin (4970), p53 (9282), GM130 (12480), EEA1 (3288), ubiquitin (43124), and MYOIIA (3403) from Cell Signaling Technology; against ACBD3 (sc-101277), GRASP65 (sc-374423), and p53 (sc-126) from Santa Cruz Biotechnology; against calnexin (ab22595) and RAB11 (ab95375) from Abcam; and against RNA polymerase II (05-623, clone CTD4H8) from MilliporeSigma.

Techniques: Mutagenesis, Transfection, Injection, Expressing

(A) Invasion assays of H1299 cells in Boyden chambers performed after 24 hours of treatment with conditioned medium (CM) from G55-deficient or G55-replete cells. (B) Anchorage-dependent colony formation assays performed on G55-deficient (shG55) or G55-replete (shCTL) H2122 cells after treatment for 10 days with or without (–) CM from G55-deficient or G55-replete cells. (C) Volcano plot of proteins identified by LC-MS analysis of CM collected from H1299 cells (shG55 or shCTL). Proteins (dots) plotted by P value (y axis) and fold change (x axis). Fold change = 1.4, P = 0.05 indicated (vertical and horizontal dotted lines, respectively). Proteins of interest are labeled. (D) WB analysis of CM samples. Mean densitometric values of triplicate CM samples relative to control (siCTL) (bar graph). (E and F) Anchorage-independent colony formation (E) and cell migration and invasion (F) assays. Results expressed relative to control (siCTL). (G) Orthotopic lung tumor size and contralateral lung metastasis numbers at necropsy 5 weeks after injecting 1 × 106 tumor cells. (H–J) WB analysis of triplicate CM samples from parental and TP53-KO A549 cells (H) or H1299 cells subjected to ectopic expression of p53 (I) or miR-34a (J). Densitometric values relative to controls (bar graphs). Values are mean ± SD. n = 3, unless otherwise indicated. P values: ANOVA (A, B, and D–G), 2-sided t test (all others).

Journal: The Journal of Clinical Investigation

Article Title: A protumorigenic secretory pathway activated by p53 deficiency in lung adenocarcinoma

doi: 10.1172/JCI137186

Figure Lengend Snippet: (A) Invasion assays of H1299 cells in Boyden chambers performed after 24 hours of treatment with conditioned medium (CM) from G55-deficient or G55-replete cells. (B) Anchorage-dependent colony formation assays performed on G55-deficient (shG55) or G55-replete (shCTL) H2122 cells after treatment for 10 days with or without (–) CM from G55-deficient or G55-replete cells. (C) Volcano plot of proteins identified by LC-MS analysis of CM collected from H1299 cells (shG55 or shCTL). Proteins (dots) plotted by P value (y axis) and fold change (x axis). Fold change = 1.4, P = 0.05 indicated (vertical and horizontal dotted lines, respectively). Proteins of interest are labeled. (D) WB analysis of CM samples. Mean densitometric values of triplicate CM samples relative to control (siCTL) (bar graph). (E and F) Anchorage-independent colony formation (E) and cell migration and invasion (F) assays. Results expressed relative to control (siCTL). (G) Orthotopic lung tumor size and contralateral lung metastasis numbers at necropsy 5 weeks after injecting 1 × 106 tumor cells. (H–J) WB analysis of triplicate CM samples from parental and TP53-KO A549 cells (H) or H1299 cells subjected to ectopic expression of p53 (I) or miR-34a (J). Densitometric values relative to controls (bar graphs). Values are mean ± SD. n = 3, unless otherwise indicated. P values: ANOVA (A, B, and D–G), 2-sided t test (all others).

Article Snippet: We purchased primary antibodies against G55 (NB100-74431) from Novus Biologicals; against GM130 (560066) from BD Transduction Laboratories; against G45 (OAAN01805) from Aviva Systems Biology; against GOLPH3 (ab98023) from Abcam; against IGFBP2 (11065-3-AP), SPP1 (22952-1-AP), MYO18A (14611-1-AP), MAOA (10539-1-AP), and RAB6A (10187-2-AP) from Proteintech; against α-tubulin (T9026), EGFP (G6539) from MilliporeSigma; against PARP-1 (9542), hemagglutinin (3724 and 2367), β-actin (4970), p53 (9282), GM130 (12480), EEA1 (3288), ubiquitin (43124), and MYOIIA (3403) from Cell Signaling Technology; against ACBD3 (sc-101277), GRASP65 (sc-374423), and p53 (sc-126) from Santa Cruz Biotechnology; against calnexin (ab22595) and RAB11 (ab95375) from Abcam; and against RNA polymerase II (05-623, clone CTD4H8) from MilliporeSigma.

Techniques: Liquid Chromatography with Mass Spectroscopy, Labeling, Migration, Expressing

(A and B) Co-IP of G55 and GOLGIN45 (G45) (A) or GRASP65 (G65) and GM130 (B). IP/WB analysis of H1299 cells transfected with GFP-tagged G55 (A) or G65 (B) and treated for 2 days with 10 μM GRASPIN or vehicle (DMSO). Whole cell lysates (input). (C) Cell proliferation after 4 days of GRASPIN treatment. Results relative to lowest GRASPIN dose in H1299_shCTL cells. n = 5. (D) Anchorage-independent colony formation. Colonies quantified after 14 days of GRASPIN or DMSO treatment. Results relative to controls (shCTL/DMSO). (E) Anchorage-dependent colony formation after 10 days of GRASPIN treatment. Results expressed relative to vehicle (0 μM). (F) The percentage reductions in E analyzed based on TP53 mutational status. TP53-WT, TP53-deleted (null), or TP53-missense-mutant (MT). (G) WB analysis of CM samples from indicated cells after 2 days of 10 μM GRASPIN or vehicle treatment. Densitometric values relative to DMSO (bar graph). (H) Co-IP of G55 and G45 in lysates of subcutaneous 344SQ tumors. Mice treated with GRASPIN or vehicle (DMSO) for 4 weeks beginning 7 days after tumor cell injection. (I and J) Tumor measurements on mice in H during treatment (I) and at necropsy (J). (K) Orthotopic lung tumor size and contralateral lung metastasis numbers in nu/nu mice treated for 3 weeks with 50 mg/kg GRASPIN or vehicle beginning 2 weeks after H1299 cell injection. (L) PDX tumor volumes in mice treated with 50 mg/kg GRASPIN or vehicle expressed as fold change relative to day 0. n = 10. (M) Orthotopic lung tumor size and contralateral lung metastasis numbers in nu/nu mice treated for 3 weeks with 50 mg/kg GRASPIN or vehicle beginning 2 weeks after A549 cell injection. n = 10. Results represent mean ± SD. n = 3, unless otherwise indicated. P values: ANOVA (E, I, and J), 2-sided t test (all others).

Journal: The Journal of Clinical Investigation

Article Title: A protumorigenic secretory pathway activated by p53 deficiency in lung adenocarcinoma

doi: 10.1172/JCI137186

Figure Lengend Snippet: (A and B) Co-IP of G55 and GOLGIN45 (G45) (A) or GRASP65 (G65) and GM130 (B). IP/WB analysis of H1299 cells transfected with GFP-tagged G55 (A) or G65 (B) and treated for 2 days with 10 μM GRASPIN or vehicle (DMSO). Whole cell lysates (input). (C) Cell proliferation after 4 days of GRASPIN treatment. Results relative to lowest GRASPIN dose in H1299_shCTL cells. n = 5. (D) Anchorage-independent colony formation. Colonies quantified after 14 days of GRASPIN or DMSO treatment. Results relative to controls (shCTL/DMSO). (E) Anchorage-dependent colony formation after 10 days of GRASPIN treatment. Results expressed relative to vehicle (0 μM). (F) The percentage reductions in E analyzed based on TP53 mutational status. TP53-WT, TP53-deleted (null), or TP53-missense-mutant (MT). (G) WB analysis of CM samples from indicated cells after 2 days of 10 μM GRASPIN or vehicle treatment. Densitometric values relative to DMSO (bar graph). (H) Co-IP of G55 and G45 in lysates of subcutaneous 344SQ tumors. Mice treated with GRASPIN or vehicle (DMSO) for 4 weeks beginning 7 days after tumor cell injection. (I and J) Tumor measurements on mice in H during treatment (I) and at necropsy (J). (K) Orthotopic lung tumor size and contralateral lung metastasis numbers in nu/nu mice treated for 3 weeks with 50 mg/kg GRASPIN or vehicle beginning 2 weeks after H1299 cell injection. (L) PDX tumor volumes in mice treated with 50 mg/kg GRASPIN or vehicle expressed as fold change relative to day 0. n = 10. (M) Orthotopic lung tumor size and contralateral lung metastasis numbers in nu/nu mice treated for 3 weeks with 50 mg/kg GRASPIN or vehicle beginning 2 weeks after A549 cell injection. n = 10. Results represent mean ± SD. n = 3, unless otherwise indicated. P values: ANOVA (E, I, and J), 2-sided t test (all others).

Article Snippet: We purchased primary antibodies against G55 (NB100-74431) from Novus Biologicals; against GM130 (560066) from BD Transduction Laboratories; against G45 (OAAN01805) from Aviva Systems Biology; against GOLPH3 (ab98023) from Abcam; against IGFBP2 (11065-3-AP), SPP1 (22952-1-AP), MYO18A (14611-1-AP), MAOA (10539-1-AP), and RAB6A (10187-2-AP) from Proteintech; against α-tubulin (T9026), EGFP (G6539) from MilliporeSigma; against PARP-1 (9542), hemagglutinin (3724 and 2367), β-actin (4970), p53 (9282), GM130 (12480), EEA1 (3288), ubiquitin (43124), and MYOIIA (3403) from Cell Signaling Technology; against ACBD3 (sc-101277), GRASP65 (sc-374423), and p53 (sc-126) from Santa Cruz Biotechnology; against calnexin (ab22595) and RAB11 (ab95375) from Abcam; and against RNA polymerase II (05-623, clone CTD4H8) from MilliporeSigma.

Techniques: Co-Immunoprecipitation Assay, Transfection, Mutagenesis, Injection

(A and B) WB analysis of triplicate CM samples from H1299 cells treated for 16 hours with 100 nM wortmannin (Wort) or 5 mM 3-methyladenine (3-MA) (A) or 1 μg/mL brefeldin A (BFA) (B). (C) WB analysis to confirm GOLPH3 depletion in H2122 cells transfected with GOLPH3 siRNAs (siGOL#1, #2). (D and E) WB analysis of triplicate CM samples from H2122 cells transfected with siRNAs against GOLPH3. Densitometric values relative to siCTL (E). (F) Kaplan-Meier analysis of the TCGA LUAD cohort (n = 477 patients) scored on the basis of G45 mRNA levels above (high) or below (low) the median value. (G) WB analysis of G45 protein levels in cells transfected with siRNAs against G45 (siG45) or control siRNA (siCTL). (H) WB analysis of triplicate CM samples from cells in G. Densitometric values relative to siCTL. (I) Anchorage-independent colony formation assays in soft agar. G45-deficient (shG45) or G45-replete (shCTL) H1299 cells cultured with or without (–) CM from shG45- or shCTL-transfected H1299 cells. (J) WB analysis to confirm G45 depletion or ectopic G55 expression. 344P cells transfected with G55 or shG45 expression vectors. Empty vectors indicated by (–). Arrow indicates the G45-specific band. (K) Primary tumor weights and lung metastasis numbers in syngeneic, immunocompetent mice determined at necropsy 5 weeks after subcutaneous injection of 1 × 106 cells in J. Results represent mean ± SD. n = 3, unless otherwise indicated. P values: log-rank test (F), ANOVA (all others).

Journal: The Journal of Clinical Investigation

Article Title: A protumorigenic secretory pathway activated by p53 deficiency in lung adenocarcinoma

doi: 10.1172/JCI137186

Figure Lengend Snippet: (A and B) WB analysis of triplicate CM samples from H1299 cells treated for 16 hours with 100 nM wortmannin (Wort) or 5 mM 3-methyladenine (3-MA) (A) or 1 μg/mL brefeldin A (BFA) (B). (C) WB analysis to confirm GOLPH3 depletion in H2122 cells transfected with GOLPH3 siRNAs (siGOL#1, #2). (D and E) WB analysis of triplicate CM samples from H2122 cells transfected with siRNAs against GOLPH3. Densitometric values relative to siCTL (E). (F) Kaplan-Meier analysis of the TCGA LUAD cohort (n = 477 patients) scored on the basis of G45 mRNA levels above (high) or below (low) the median value. (G) WB analysis of G45 protein levels in cells transfected with siRNAs against G45 (siG45) or control siRNA (siCTL). (H) WB analysis of triplicate CM samples from cells in G. Densitometric values relative to siCTL. (I) Anchorage-independent colony formation assays in soft agar. G45-deficient (shG45) or G45-replete (shCTL) H1299 cells cultured with or without (–) CM from shG45- or shCTL-transfected H1299 cells. (J) WB analysis to confirm G45 depletion or ectopic G55 expression. 344P cells transfected with G55 or shG45 expression vectors. Empty vectors indicated by (–). Arrow indicates the G45-specific band. (K) Primary tumor weights and lung metastasis numbers in syngeneic, immunocompetent mice determined at necropsy 5 weeks after subcutaneous injection of 1 × 106 cells in J. Results represent mean ± SD. n = 3, unless otherwise indicated. P values: log-rank test (F), ANOVA (all others).

Article Snippet: We purchased primary antibodies against G55 (NB100-74431) from Novus Biologicals; against GM130 (560066) from BD Transduction Laboratories; against G45 (OAAN01805) from Aviva Systems Biology; against GOLPH3 (ab98023) from Abcam; against IGFBP2 (11065-3-AP), SPP1 (22952-1-AP), MYO18A (14611-1-AP), MAOA (10539-1-AP), and RAB6A (10187-2-AP) from Proteintech; against α-tubulin (T9026), EGFP (G6539) from MilliporeSigma; against PARP-1 (9542), hemagglutinin (3724 and 2367), β-actin (4970), p53 (9282), GM130 (12480), EEA1 (3288), ubiquitin (43124), and MYOIIA (3403) from Cell Signaling Technology; against ACBD3 (sc-101277), GRASP65 (sc-374423), and p53 (sc-126) from Santa Cruz Biotechnology; against calnexin (ab22595) and RAB11 (ab95375) from Abcam; and against RNA polymerase II (05-623, clone CTD4H8) from MilliporeSigma.

Techniques: Transfection, Cell Culture, Expressing, Injection

(A) Co-IP of G55 and G45. IP/WB assays on H1299 cells cotransfected with GFP-tagged G55 (GFP-G55) and HA-tagged WT or mutant (L400R) G45 (HA-G45). (B) WB analysis of H1299 cells cotransfected with G45 shRNA and WT or mutant (L400R) G45 (G45). Control vectors indicated by (–). Arrow indicates endogenous G45. (C) Anchorage-independent colony formation using cells in B. (D) Co-IP of G55 and G45. IP/WB analysis of H1299 cells cotransfected with HA-tagged G45 and GFP-tagged WT or mutant (G97D) G55. (E) WB analysis of H1299 cells cotransfected with G55 shRNA (shG55) and WT or mutant (G97D) G55. Endogenous (G55) and ectopic (G55-EGFP) G55. (F) WB analysis of CM samples from cells in E. (G) Anchorage-independent colony formation assays on cells in E. (H) Primary tumor weights and lung metastasis numbers at necropsy 5 weeks after subcutaneously injecting 1 × 106 344SQ cells into syngeneic, immunocompetent mice. Transfectants generated using the same vectors described in E. (I) Co-IP of G55 and G45. IP/WB assays on H1299 cells cotransfected with GFP-tagged G55 and a G45 peptide that blocks G55/G45 interactions (G45 BP). Empty vector indicated by (–). Arrow indicates endogenous G45. (J) Anchorage-independent colony formation using H1299 cells or 344SQ cells transfected with G45 BP or empty vector (–). (K) Primary tumor weights and lung metastasis numbers at necropsy 5 weeks after subcutaneous injection of 344SQ cells in J. (L) Kaplan-Meier analysis of nu/nu mice bearing orthotopic lung tumors generated by H1299 cells in J. Results represent mean ± SD. n = 3, unless otherwise indicated. P values: ANOVA (C, G, and H), log-rank test (L), 2-sided t test (all others).

Journal: The Journal of Clinical Investigation

Article Title: A protumorigenic secretory pathway activated by p53 deficiency in lung adenocarcinoma

doi: 10.1172/JCI137186

Figure Lengend Snippet: (A) Co-IP of G55 and G45. IP/WB assays on H1299 cells cotransfected with GFP-tagged G55 (GFP-G55) and HA-tagged WT or mutant (L400R) G45 (HA-G45). (B) WB analysis of H1299 cells cotransfected with G45 shRNA and WT or mutant (L400R) G45 (G45). Control vectors indicated by (–). Arrow indicates endogenous G45. (C) Anchorage-independent colony formation using cells in B. (D) Co-IP of G55 and G45. IP/WB analysis of H1299 cells cotransfected with HA-tagged G45 and GFP-tagged WT or mutant (G97D) G55. (E) WB analysis of H1299 cells cotransfected with G55 shRNA (shG55) and WT or mutant (G97D) G55. Endogenous (G55) and ectopic (G55-EGFP) G55. (F) WB analysis of CM samples from cells in E. (G) Anchorage-independent colony formation assays on cells in E. (H) Primary tumor weights and lung metastasis numbers at necropsy 5 weeks after subcutaneously injecting 1 × 106 344SQ cells into syngeneic, immunocompetent mice. Transfectants generated using the same vectors described in E. (I) Co-IP of G55 and G45. IP/WB assays on H1299 cells cotransfected with GFP-tagged G55 and a G45 peptide that blocks G55/G45 interactions (G45 BP). Empty vector indicated by (–). Arrow indicates endogenous G45. (J) Anchorage-independent colony formation using H1299 cells or 344SQ cells transfected with G45 BP or empty vector (–). (K) Primary tumor weights and lung metastasis numbers at necropsy 5 weeks after subcutaneous injection of 344SQ cells in J. (L) Kaplan-Meier analysis of nu/nu mice bearing orthotopic lung tumors generated by H1299 cells in J. Results represent mean ± SD. n = 3, unless otherwise indicated. P values: ANOVA (C, G, and H), log-rank test (L), 2-sided t test (all others).

Article Snippet: We purchased primary antibodies against G55 (NB100-74431) from Novus Biologicals; against GM130 (560066) from BD Transduction Laboratories; against G45 (OAAN01805) from Aviva Systems Biology; against GOLPH3 (ab98023) from Abcam; against IGFBP2 (11065-3-AP), SPP1 (22952-1-AP), MYO18A (14611-1-AP), MAOA (10539-1-AP), and RAB6A (10187-2-AP) from Proteintech; against α-tubulin (T9026), EGFP (G6539) from MilliporeSigma; against PARP-1 (9542), hemagglutinin (3724 and 2367), β-actin (4970), p53 (9282), GM130 (12480), EEA1 (3288), ubiquitin (43124), and MYOIIA (3403) from Cell Signaling Technology; against ACBD3 (sc-101277), GRASP65 (sc-374423), and p53 (sc-126) from Santa Cruz Biotechnology; against calnexin (ab22595) and RAB11 (ab95375) from Abcam; and against RNA polymerase II (05-623, clone CTD4H8) from MilliporeSigma.

Techniques: Co-Immunoprecipitation Assay, Mutagenesis, shRNA, Generated, Plasmid Preparation, Transfection, Injection

(A and B) Pearson’s correlation of G55, G45, MYOIIA, and MYO18A in LUAD (A) and pan-cancer (B) TCGA cohorts. MYO18A included as negative control. (C and D) G45 (C) and MYOIIA (D) mRNA levels in tumors (dots) annotated on the basis of somatic DNA mutations in the TCGA pan-cancer cohort. Sample sizes indicated (n). Tumor types color-coded. “Unaligned” samples did not correlate with any of the indicated genetic mutations. (E and F) Kaplan-Meier analysis of TCGA cohorts on the basis of a 3-gene expression signature (G55, G45, MYOIIA). For the LUAD cohort (E), tumors in the top third (high), middle third (other), and bottom third (low) were compared. For the pan-cancer cohort (F), tumors in the top third (high) were compared with tumors in the middle and bottom third (other). (G) qPCR analysis of MYOIIA mRNA levels in A549 cells cotransfected with shp53 or shCTL and miR-34 or miR-NC. Results expressed relative to controls (shCTL/miR-NC). (H) qPCR analysis of MYOIIA mRNA levels in H1299 cells cotransfected with p53 or empty (Vec) expression vector and antagomir-34a (anti-34a) or control (anti-NC). Results expressed relative to controls (Vec/anti-NC). (I) WB analysis of H1299 cells and H2122 cells transfected with miR-34a, miR-200a, or control (miR-NC). Densitometric values relative to miR-NC. (J) Reporter assays on H1299 cells cotransfected with miR-34a or miR-NC and reporters containing MYOIIA 3′-UTRs with intact (WT) or mutant (MT) miR-34a binding sites. A MYO18A 3′-UTR reporter that lacks miR-34a binding sites included as a control. Values expressed relative to miR-NC. (K) Schematic illustration of our working model. Results represent mean ± SD. n = 3, unless otherwise indicated. P values: Pearson’s correlation (A and B), ANOVA (C, D, G, and H), univariate Cox (E and F), 2-way t test (J).

Journal: The Journal of Clinical Investigation

Article Title: A protumorigenic secretory pathway activated by p53 deficiency in lung adenocarcinoma

doi: 10.1172/JCI137186

Figure Lengend Snippet: (A and B) Pearson’s correlation of G55, G45, MYOIIA, and MYO18A in LUAD (A) and pan-cancer (B) TCGA cohorts. MYO18A included as negative control. (C and D) G45 (C) and MYOIIA (D) mRNA levels in tumors (dots) annotated on the basis of somatic DNA mutations in the TCGA pan-cancer cohort. Sample sizes indicated (n). Tumor types color-coded. “Unaligned” samples did not correlate with any of the indicated genetic mutations. (E and F) Kaplan-Meier analysis of TCGA cohorts on the basis of a 3-gene expression signature (G55, G45, MYOIIA). For the LUAD cohort (E), tumors in the top third (high), middle third (other), and bottom third (low) were compared. For the pan-cancer cohort (F), tumors in the top third (high) were compared with tumors in the middle and bottom third (other). (G) qPCR analysis of MYOIIA mRNA levels in A549 cells cotransfected with shp53 or shCTL and miR-34 or miR-NC. Results expressed relative to controls (shCTL/miR-NC). (H) qPCR analysis of MYOIIA mRNA levels in H1299 cells cotransfected with p53 or empty (Vec) expression vector and antagomir-34a (anti-34a) or control (anti-NC). Results expressed relative to controls (Vec/anti-NC). (I) WB analysis of H1299 cells and H2122 cells transfected with miR-34a, miR-200a, or control (miR-NC). Densitometric values relative to miR-NC. (J) Reporter assays on H1299 cells cotransfected with miR-34a or miR-NC and reporters containing MYOIIA 3′-UTRs with intact (WT) or mutant (MT) miR-34a binding sites. A MYO18A 3′-UTR reporter that lacks miR-34a binding sites included as a control. Values expressed relative to miR-NC. (K) Schematic illustration of our working model. Results represent mean ± SD. n = 3, unless otherwise indicated. P values: Pearson’s correlation (A and B), ANOVA (C, D, G, and H), univariate Cox (E and F), 2-way t test (J).

Article Snippet: We purchased primary antibodies against G55 (NB100-74431) from Novus Biologicals; against GM130 (560066) from BD Transduction Laboratories; against G45 (OAAN01805) from Aviva Systems Biology; against GOLPH3 (ab98023) from Abcam; against IGFBP2 (11065-3-AP), SPP1 (22952-1-AP), MYO18A (14611-1-AP), MAOA (10539-1-AP), and RAB6A (10187-2-AP) from Proteintech; against α-tubulin (T9026), EGFP (G6539) from MilliporeSigma; against PARP-1 (9542), hemagglutinin (3724 and 2367), β-actin (4970), p53 (9282), GM130 (12480), EEA1 (3288), ubiquitin (43124), and MYOIIA (3403) from Cell Signaling Technology; against ACBD3 (sc-101277), GRASP65 (sc-374423), and p53 (sc-126) from Santa Cruz Biotechnology; against calnexin (ab22595) and RAB11 (ab95375) from Abcam; and against RNA polymerase II (05-623, clone CTD4H8) from MilliporeSigma.

Techniques: Negative Control, Expressing, Plasmid Preparation, Transfection, Mutagenesis, Binding Assay

(A and B) Co-IP of G45 and myosin IIA (MYOIIA). IP/WB assays on H1299 cells cotransfected with GFP-tagged MYOIIA and HA-tagged G45 (A). H1299 cells transfected with WT or mutant (ΔN) HA-tagged G45 lacking N-terminal peptides (B). (C) WB analysis of total (–) and fractionated (+) H1299 cell lysates to confirm enrichment in “vesicle” and “Golgi” fractions on the basis of organelle markers (right). (D) WB analysis of RAB6A, IGFBP2, and SPP1 in Golgi- or vesicle-enriched fractions or total lysates (–) isolated from H1299 cells transfected with siRNA against G55, G45, or MYOIIA. (E) Densitometric analysis of results in D expressed relative to siCTL controls. (F) Contrast-adjusted confocal micrographs. RAB6A vesicles (red arrows) and unfissioned RAB6A tubules (blue arrows) emerge from the Golgi (green arrows). Cell boundary (dotted lines). Size bars: 10 μm. (G–J) Quantification of vesicles (G and H) and unfissioned tubules (I and J) per cell (dots). (K and L) WB analysis of CM samples isolated from H1299 cells and H2122 cells transfected with siRNAs against MYOIIA (K) or RAB6A (L). Densitometric values expressed relative to siCTL (bar graphs). Results represent mean ± SD. n = 3, unless otherwise indicated. P values: ANOVA (E and K), 2-way t test (all others).

Journal: The Journal of Clinical Investigation

Article Title: A protumorigenic secretory pathway activated by p53 deficiency in lung adenocarcinoma

doi: 10.1172/JCI137186

Figure Lengend Snippet: (A and B) Co-IP of G45 and myosin IIA (MYOIIA). IP/WB assays on H1299 cells cotransfected with GFP-tagged MYOIIA and HA-tagged G45 (A). H1299 cells transfected with WT or mutant (ΔN) HA-tagged G45 lacking N-terminal peptides (B). (C) WB analysis of total (–) and fractionated (+) H1299 cell lysates to confirm enrichment in “vesicle” and “Golgi” fractions on the basis of organelle markers (right). (D) WB analysis of RAB6A, IGFBP2, and SPP1 in Golgi- or vesicle-enriched fractions or total lysates (–) isolated from H1299 cells transfected with siRNA against G55, G45, or MYOIIA. (E) Densitometric analysis of results in D expressed relative to siCTL controls. (F) Contrast-adjusted confocal micrographs. RAB6A vesicles (red arrows) and unfissioned RAB6A tubules (blue arrows) emerge from the Golgi (green arrows). Cell boundary (dotted lines). Size bars: 10 μm. (G–J) Quantification of vesicles (G and H) and unfissioned tubules (I and J) per cell (dots). (K and L) WB analysis of CM samples isolated from H1299 cells and H2122 cells transfected with siRNAs against MYOIIA (K) or RAB6A (L). Densitometric values expressed relative to siCTL (bar graphs). Results represent mean ± SD. n = 3, unless otherwise indicated. P values: ANOVA (E and K), 2-way t test (all others).

Article Snippet: We purchased primary antibodies against G55 (NB100-74431) from Novus Biologicals; against GM130 (560066) from BD Transduction Laboratories; against G45 (OAAN01805) from Aviva Systems Biology; against GOLPH3 (ab98023) from Abcam; against IGFBP2 (11065-3-AP), SPP1 (22952-1-AP), MYO18A (14611-1-AP), MAOA (10539-1-AP), and RAB6A (10187-2-AP) from Proteintech; against α-tubulin (T9026), EGFP (G6539) from MilliporeSigma; against PARP-1 (9542), hemagglutinin (3724 and 2367), β-actin (4970), p53 (9282), GM130 (12480), EEA1 (3288), ubiquitin (43124), and MYOIIA (3403) from Cell Signaling Technology; against ACBD3 (sc-101277), GRASP65 (sc-374423), and p53 (sc-126) from Santa Cruz Biotechnology; against calnexin (ab22595) and RAB11 (ab95375) from Abcam; and against RNA polymerase II (05-623, clone CTD4H8) from MilliporeSigma.

Techniques: Co-Immunoprecipitation Assay, Transfection, Mutagenesis, Isolation

PLCD1 ectopic expression inhibited oncogenic signaling. a Experimentally expressed PLCD1 in 293 T cells was confirmed by Western blot. b Activity of several oncogenic signaling reporters were evaluated using dual-luciferase reporters assay in 293 T cells. b TCF activity and CCND1 , c-Myc and MMP7 promoter reporter activities in PLCD1 -overexpressing A498 (upper) and HH244 (bottom) cells. d Western blot analyses of total β-catenin, p-β-catenin (Ser552), c-Myc, active β-catenin, cyclinD1, MMP7, PLCD1 and GAPDH. e Western blot analyses of EGFR, ERK1/2, Src, FAK and PLCD1 , with GAPDH as internal control. Histograms indicated quantification of the phosphorylated protein normalized to corresponding total protein level. f Schematic diagram of roles and mechanisms of PLCD1 in RCC tumorigenesis

Journal: Clinical Epigenetics

Article Title: Phospholipase C delta 1 inhibits WNT/β‐catenin and EGFR-FAK-ERK signaling and is disrupted by promoter CpG methylation in renal cell carcinoma

doi: 10.1186/s13148-023-01448-2

Figure Lengend Snippet: PLCD1 ectopic expression inhibited oncogenic signaling. a Experimentally expressed PLCD1 in 293 T cells was confirmed by Western blot. b Activity of several oncogenic signaling reporters were evaluated using dual-luciferase reporters assay in 293 T cells. b TCF activity and CCND1 , c-Myc and MMP7 promoter reporter activities in PLCD1 -overexpressing A498 (upper) and HH244 (bottom) cells. d Western blot analyses of total β-catenin, p-β-catenin (Ser552), c-Myc, active β-catenin, cyclinD1, MMP7, PLCD1 and GAPDH. e Western blot analyses of EGFR, ERK1/2, Src, FAK and PLCD1 , with GAPDH as internal control. Histograms indicated quantification of the phosphorylated protein normalized to corresponding total protein level. f Schematic diagram of roles and mechanisms of PLCD1 in RCC tumorigenesis

Article Snippet: Antibodies used are: anti-mouse IgG-HRP (DAKO, P0161), anti-rabbit IgG-HRP (DAKO, P0448), GAPDH (Millipore, MAB374), Anti-V5-Tag (Invitrogen, R96025), E-cadherin (CST, 4065), vimentin (Sigma: V6630), Twist (Santa Cruz, sc-15393), MMP7 (Thermo Fisher, MS-813-P0), Total EGFR (CST: 54,359), p-EGFR (CST: 3777), Total FAK (CST: 71,433), p-FAK (Tyr397) (CST: 8556), Total SRC (CST: 2191), p-SRC (CST: 59,548), Total β-catenin (CST: 59,548), active β-catenin (Millipore, 05,665), p-β-catenin (Ser552) (CST: 5651), c-Myc (CST: 18,583), cyclinD1 (CST: 55,506), p-ERK1/2 (CST: 9101), Total ERK1/2 (CST: 4695), Cleaved-Caspase3 (CST: 9661), Cleaved-PARP (CST: 9541), Caspase 3 (CST:9504), PARP (CST: 9532), Bax (CST:2772), Bcl-2 (CST: 2872).

Techniques: Expressing, Western Blot, Activity Assay, Luciferase

DKI mice have reduced skeletal muscle AMPK α and β2 content but intact AMPK, ACC and TBC1D1 signaling in response to a maximal running test at a 5° incline versus WT. Skeletal muscles were collected from rested and maximally exercised male WT and DKI mice, and phosphorylation of AMPK and downstream substrates was assessed using Western blotting. (A) Representative immunoblots of p-T172 and total AMPK, p-S182 and total AMPK β, p-S79 and total acetyl-CoA carboxylase (ACC) and p-S660 and total TBC1 domain family member 1 (TBC1D1) with representative stain free image. Quantified relative (B) AMPK p-T172, (C) AMPK β p-S182, (D) ACC p-S79, and (E) TBC1D1 p-S660; (F) Total AMPK α; (G) Total AMPK β. Male mice, 17–20 weeks, n = 6–8. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Journal: Frontiers in Physiology

Article Title: Disrupting AMPK-Glycogen Binding in Mice Increases Carbohydrate Utilization and Reduces Exercise Capacity

doi: 10.3389/fphys.2022.859246

Figure Lengend Snippet: DKI mice have reduced skeletal muscle AMPK α and β2 content but intact AMPK, ACC and TBC1D1 signaling in response to a maximal running test at a 5° incline versus WT. Skeletal muscles were collected from rested and maximally exercised male WT and DKI mice, and phosphorylation of AMPK and downstream substrates was assessed using Western blotting. (A) Representative immunoblots of p-T172 and total AMPK, p-S182 and total AMPK β, p-S79 and total acetyl-CoA carboxylase (ACC) and p-S660 and total TBC1 domain family member 1 (TBC1D1) with representative stain free image. Quantified relative (B) AMPK p-T172, (C) AMPK β p-S182, (D) ACC p-S79, and (E) TBC1D1 p-S660; (F) Total AMPK α; (G) Total AMPK β. Male mice, 17–20 weeks, n = 6–8. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Antibodies against total AMPK α (2532), phospho-AMPK T172 (2531), total AMPK β (4150), phospho-AMPK β1 S182 (4186), total ACC (3662), phospho-ACC S79 (11,818), GLUT4 (2213), total TBC1D1 (66,433), phospho-TBC1D1 S660 (6928) and horseradish peroxidase-conjugated anti-rabbit (7074) and anti-mouse (7076) IgG secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, United States).

Techniques: Western Blot, Staining

Specifics of the four exercise protocols used and corresponding measures.

Journal: Frontiers in Physiology

Article Title: Disrupting AMPK-Glycogen Binding in Mice Increases Carbohydrate Utilization and Reduces Exercise Capacity

doi: 10.3389/fphys.2022.859246

Figure Lengend Snippet: Specifics of the four exercise protocols used and corresponding measures.

Article Snippet: Antibodies against total AMPK α (2532), phospho-AMPK T172 (2531), total AMPK β (4150), phospho-AMPK β1 S182 (4186), total ACC (3662), phospho-ACC S79 (11,818), GLUT4 (2213), total TBC1D1 (66,433), phospho-TBC1D1 S660 (6928) and horseradish peroxidase-conjugated anti-rabbit (7074) and anti-mouse (7076) IgG secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, United States).

Techniques: