Journal: Journal of Biological Chemistry

Article Title: Activation of Protein Kinase Cζ by Peroxynitrite Regulates LKB1-dependent AMP-activated Protein Kinase in Cultured Endothelial Cells

doi: 10.1074/jbc.m511178200

Figure Lengend Snippet: FIGURE 5. LKB1 is required for ONOO-depend- ent AMPK activation. a, ONOO activates AMPK in BAEC, but not LKB1-deficient HeLa S3 cells. The blot is representative of at least three blots from at least three independent assays. b, ONOO did not activate AMPK in LKB1-deficient HeLa-S3. Conflu- ent HeLa-S3 cells were exposed to ONOO, and AMPK activity was assayed as described under “ExperimentalProcedures”(n5).c,ONOOdoes not alter LKB1 activity. LKB1 was first immunopre- cipitated from BAEC and then exposed to ONOO. LKB1 activity was assayed by including recombi- nant AMPK as described under “Experimental Pro- cedures” (n 5).

Article Snippet: Antibodies against phospho-AMPK (Thr172), AMPK- , phospho-eNOS (Ser1177), phosphor-LKB1 (Ser428, Ser334, and Thr189), LKB1 (for Western blot), PKC , phospho-PKC (Thr410–403), PKC / , and phospho-PKC / (Thr638/641) were obtained from Cell Signaling Inc. (Beverly, MA).

Techniques: Activation Assay, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Activation of Protein Kinase Cζ by Peroxynitrite Regulates LKB1-dependent AMP-activated Protein Kinase in Cultured Endothelial Cells

doi: 10.1074/jbc.m511178200

Figure Lengend Snippet: FIGURE 6. ONOO via PKC enhances the co-immunoprecipitation of LKB1 and AMPK. a, ONOO increases the association of LKB1 and AMPK. LKB1 or AMPK were immuno- precipitated (IP) from BAEC and AMPK or LKB1 detected in Western blots (WB). The blot is representative of five blots from five independent experiments. b, inhibition of PKC attenuates ONOO-enhanced association of LKB1 with AMPK. LKB1 was immunoprecipitated and stained for AMPK in Western blots. Of note is that ONOO increased LKB1 associationwithAMPKinBAEC,whichwassensitivetoPKC-PS.Theblotisrepresentativeoffiveblotsfromfiveindependentexperiments.c,ONOOincreasesLKB1-associatedAMPK activity. LKB1 were immunoprecipitated as described above. AMPK activity was assayed by [32P]ATP phosphorylation of the SAMS peptide, as described under “Experimental Procedures” (n 5; , p 0.05 control versus ONOO; , p 0.05 ONOO versus ONOO plus PKC-PS. d, ONOO increased the co-immunoprecipitation of LKB1 with MARK-3. The lower panel is the results of MARK-3 staining in LKB1 immunoprecipitates (n 3; , p 0.05). Of note is that ONOO slightly increased the co-immunoprecipitation of LKB1 with MARK-3. However, PKC does not alter ONOO-enhanced co-immunoprecipitation of LKB1 with MARK-3. The blot is representative of three blots from three individual experiments.

Article Snippet: Antibodies against phospho-AMPK (Thr172), AMPK- , phospho-eNOS (Ser1177), phosphor-LKB1 (Ser428, Ser334, and Thr189), LKB1 (for Western blot), PKC , phospho-PKC (Thr410–403), PKC / , and phospho-PKC / (Thr638/641) were obtained from Cell Signaling Inc. (Beverly, MA).

Techniques: Immunoprecipitation, Western Blot, Inhibition, Staining, Activity Assay, Phospho-proteomics, Control

Journal: Journal of Biological Chemistry

Article Title: Activation of Protein Kinase Cζ by Peroxynitrite Regulates LKB1-dependent AMP-activated Protein Kinase in Cultured Endothelial Cells

doi: 10.1074/jbc.m511178200

Figure Lengend Snippet: FIGURE 7. PKC increases the co-immunopre- cipitation of LKB1 and AMPK by enhancing the Ser428 phosphorylation of LKB1. a, inhibition of PKC attenuates ONOO-enhanced LKB1 phos- phorylation detected by the antibody against phospho-Ser/Thr. LKB1 was first immunoprecipi- tated (IP) from BAEC but Western blotted (WB) against the antibody against phosphorylated Ser/ Thr. Of note is that ONOO enhanced the Ser/Thr phosphorylation, which was attenuated by PKC- PS. The blot is representative of three blots from three independent experiments. b, PKC-depend- ent LKB1-Ser428 phosphorylation. LKB1 was first immunoprecipitated and Western blotted with antibody against phosphorylated LKB1-Ser428. Of note is that PKC-PS blocked ONOO enhanced detection of LKB1-Ser428. The blot is representa- tive of three blots from three independent exper- iments. c, ONOO does not alter the phosphoryl- ation of LKB1-Ser189 or -Ser334. The blot is representative of five blots from five independent experiments. d, recombinant PKC increases LKB1 phosphorylation at Ser428 in vitro. The blot is rep- resentative of three blots from three independent experiments.

Article Snippet: Antibodies against phospho-AMPK (Thr172), AMPK- , phospho-eNOS (Ser1177), phosphor-LKB1 (Ser428, Ser334, and Thr189), LKB1 (for Western blot), PKC , phospho-PKC (Thr410–403), PKC / , and phospho-PKC / (Thr638/641) were obtained from Cell Signaling Inc. (Beverly, MA).

Techniques: Phospho-proteomics, Inhibition, Western Blot, Immunoprecipitation, Recombinant, In Vitro

Journal: Journal of Biological Chemistry

Article Title: Activation of Protein Kinase Cζ by Peroxynitrite Regulates LKB1-dependent AMP-activated Protein Kinase in Cultured Endothelial Cells

doi: 10.1074/jbc.m511178200

Figure Lengend Snippet: FIGURE8.Pharmacologicalorgeneticalteration of PKC alters AMPK activity in cells originated from human, rat, and mouse. a, inhibition of PKC blunts ONOO-enhanced phosphorylation of AMPK-Thr172 and LKB1-Ser428 in cultured human retinal pericytes, rat vascular smooth mus- cle cells, and mouse 3T3L1 adipocytes. Of note is that ONOO increased the phosphorylation of both AMPK-Thr172 and LKB1-Ser428, whereas PKC-PS blocked the effects of ONOO. b, overex- pression of PKC-DN abolishes H/R-enhanced AMPK activity, whereas overexpression of PKC with PKC-CA enhanced the effects of hypoxia-de- oxygenating on AMPK in BAEC (n 5; , p 0.05, control versus H/R; †, p 0.05 H/R versus H/R plus PKC-DN). c, PKC-PS dose-dependently inhibits metformin-enhanced AMPK activation in BAEC (n5; ,p0.05,controlversusmetformin;†,p 0.05, metformin versus metformin plus PKC-PS). d, PMA increases the phosphorylation of LKB1 at Ser428. The blot is representative of five blots from five individual experiments. e, activation of PKC with PMA or H/R increases AMPK activation. Con- fluent BAEC were stimulated with PMA at concen- trations indicated for 30 min, and AMPK-Thr172

Article Snippet: Antibodies against phospho-AMPK (Thr172), AMPK- , phospho-eNOS (Ser1177), phosphor-LKB1 (Ser428, Ser334, and Thr189), LKB1 (for Western blot), PKC , phospho-PKC (Thr410–403), PKC / , and phospho-PKC / (Thr638/641) were obtained from Cell Signaling Inc. (Beverly, MA).

Techniques: Activity Assay, Inhibition, Phospho-proteomics, Cell Culture, Over Expression, Control, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: Activation of Protein Kinase Cζ by Peroxynitrite Regulates LKB1-dependent AMP-activated Protein Kinase in Cultured Endothelial Cells

doi: 10.1074/jbc.m511178200

Figure Lengend Snippet: FIGURE 9. Mutation of Ser428 of LKB1 into alanine (S428A) abolishes ONOO-en- hanced AMPK activation in HeLa-S3 cells. HeLa-S3 cells transfected with LKB1 wild type (WT) or LKB1 were exposed to ONOO (100 M), and AMPK activation was moni- tored in Western blots using the specific antibodies. The blot is representative of three to four blots obtained from three or four independent experiments.

Article Snippet: Antibodies against phospho-AMPK (Thr172), AMPK- , phospho-eNOS (Ser1177), phosphor-LKB1 (Ser428, Ser334, and Thr189), LKB1 (for Western blot), PKC , phospho-PKC (Thr410–403), PKC / , and phospho-PKC / (Thr638/641) were obtained from Cell Signaling Inc. (Beverly, MA).

Techniques: Mutagenesis, Activation Assay, Transfection, Western Blot

Journal: Poultry Science

Article Title: Oxidative stress induced by hydrogen peroxide promotes glycolysis by activating CaMKK/LKB1/AMPK pathway in broiler breast muscle

doi: 10.1016/j.psj.2021.101681

Figure Lengend Snippet: The primer sequences used for real-time PCR analysis.

Article Snippet: The antibodies for LKB1 (bs-3250R) and CaMKK1 (bs-6253R) were purchased from Bioss biotechnology company (Beijing).

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Poultry Science

Article Title: Oxidative stress induced by hydrogen peroxide promotes glycolysis by activating CaMKK/LKB1/AMPK pathway in broiler breast muscle

doi: 10.1016/j.psj.2021.101681

Figure Lengend Snippet: Effects of oxidative stress induced by H 2 O 2 on the content of Ca 2+ , gene and protein expression of LKB1, CaMKKα, CaMKKβ, AMPKα1, AMPKα2, HK1, PK, PFK, and GAPDH in broiler breast muscle. (A) Content of Ca 2+ in breast muscle. (b–F) Relative mRNA expression levels of LKB1, CaMKKα, CaMKKβ, AMPKα1 , and AMPKα2 . (G, H) Relative protein expression levels of HK1, PK, PFK, LKB1, CaMKK1, and Phospho-AMPK. (i) Quantification of HK1, PK, PFK, LKB1, CaMKK1 and Phospho-AMPK. Data are presented as mean ± SE (n = 6). Different letters indicate significant differences at P < 0.05.

Article Snippet: The antibodies for LKB1 (bs-3250R) and CaMKK1 (bs-6253R) were purchased from Bioss biotechnology company (Beijing).

Techniques: Expressing