Journal: Scientific Reports

Article Title: Prevention of calpain-dependent degradation of STK38 by MEKK2-mediated phosphorylation

doi: 10.1038/s41598-019-52435-8

Figure Lengend Snippet: MEKK2 inhibits heat- and calpain-mediated degradation of STK38. ( A ) COS-7 cells were transfected with human STK38-V5 alone, or with FLAG-MEKK1 or FLAG-MEKK2. Forty-eight hours after transfection, the cells were heated to 44 °C for 20 min or left untreated as controls and harvested. ( B ) COS-7 cells were transfected with human STK38-V5 alone, or with FLAG-MEKK2 (WT) or FLAG-MEKK2 (KM). Forty-eight hours after transfection, the cells were harvested. ( C ) HeLa cells were transfected with the scramble oligonucleotides control (scr) or MEKK2 -specific siRNA. Forty-eight hours after transfection, the cells were heated to 44 °C for the indicated times or left untreated as controls and harvested. ( D ) HeLa cells were transfected with FLAG-MEKK2. Forty-eight hours after transfection, the cells were heated to 44 °C for the indicated times or left untreated as controls and harvested. The MEKK2 activity was measured by immune complex kinase assay with an anti-FLAG antibody using GST-MKK4 as the substrate. ( E ) GST-STK38 was incubated with calpain I (0.07 units of calpain I in each lane) in the absence or presence of GST-active MEKK2 for 15 min at 30 °C. Cell lysates or in vitro reaction products were analysed by western blotting with the antibodies against the indicated proteins. A representative image of western blot is shown (see Supplementary Fig for corresponding full-length image). Relative levels of STK38 were determined from the western blot using Image J software. Data are presented as the mean ± standard deviation of three independent experiments. Statistical significance was determined by the Student’s t -test (** P < 0.05).

Article Snippet: For the MEKK2 kinase assay, V5-STK38 immunoprecipitates were incubated with 10 ng of active-MEKK2 (Signal Chem, Richmond, BC) in MEKK2 kinase buffer containing 0.37 MBq ml −1 [γ- 32 P] ATP at 30 °C for 15 min. For in vitro kinase assays, active MEKK2 was incubated with 1.0 μg of wild-type inactive STK38 (Signal Chem) or inactive MKK4 (Signal Chem) in MEKK2 kinase buffer containing 0.37 MBq ml −1 [γ- 32 P] ATP for 30 min at 30 °C.

Techniques: Transfection, Control, Activity Assay, Immune Complex Kinase Assay, Incubation, In Vitro, Western Blot, Software, Standard Deviation

Journal: Scientific Reports

Article Title: Prevention of calpain-dependent degradation of STK38 by MEKK2-mediated phosphorylation

doi: 10.1038/s41598-019-52435-8

Figure Lengend Snippet: Maintenance of STK38 stability requires its phosphorylation. ( A ) MEKK2 phosphorylates STK38 in vitro . GST-tagged STK38 (unactive) or MKK4 (unactive) was incubated with or without active MEKK2 in the presence of [γ- 32 P] ATP for 30 min at 30 °C. ( B , upper) Putative phosphorylation sites identified within STK38. Inactive GST-STK38 was incubated with or without active MEKK2, and the kinase reaction products were subjected to SDS-PAGE. GST-STK38 was excised and processed by tryptic cleavage for MS analysis. *T74 is endogenously phosphorylated. ( B , bottom) In vitro kinase reaction was performed by incubating active MEKK2 alone or with the V5-immunopurified wild-type STK38, STK38 (S91A), STK38 (T243A), STK38 (T270A) from the transfected 293 T cells for 15 min at 30 °C. The kinase reaction products were subjected to SDS-PAGE and then visualised by autoradiography ( 32 P, top panel) or Coomassie Brilliant Blue staining (CBB, bottom panel). ( C ) COS-7 cells were transfected with human STK38-V5 (WT) or STK38-V5 (S91A). Forty-eight hours after transfection, the cells were harvested. In vitro cleavage reaction was performed by incubating calpain I (0.07 units) with lysates (30 μg) of the transfected cells from STK38-V5 (WT) or STK38-V5 (S91A) for 30–60 min at 30 °C. Reaction products were subjected to western blotting analysis with antibodies against the indicated proteins. ( D ) COS-7 cells were transfected with human STK38-V5 (WT) or STK38-V5 (S91A). Forty-eight hours after transfection, the cells were treated as described in Fig. . Cell lysates were analysed by western blotting with antibodies against the indicated proteins. A representative image of western blot, CBB-stained gel, or autoradiography is shown (see Supplementary Fig for corresponding full-length image). Relative levels of STK38 were determined from the western blot by using Image J software. Data are presented as the mean ± standard deviation of three independent experiments. Statistical significance was determined by the Student’s t -test (* P < 0.05; ** P < 0.01).

Article Snippet: For the MEKK2 kinase assay, V5-STK38 immunoprecipitates were incubated with 10 ng of active-MEKK2 (Signal Chem, Richmond, BC) in MEKK2 kinase buffer containing 0.37 MBq ml −1 [γ- 32 P] ATP at 30 °C for 15 min. For in vitro kinase assays, active MEKK2 was incubated with 1.0 μg of wild-type inactive STK38 (Signal Chem) or inactive MKK4 (Signal Chem) in MEKK2 kinase buffer containing 0.37 MBq ml −1 [γ- 32 P] ATP for 30 min at 30 °C.

Techniques: Phospho-proteomics, In Vitro, Incubation, SDS Page, Transfection, Autoradiography, Staining, Western Blot, Software, Standard Deviation

Journal: bioRxiv

Article Title: Truncation and Motif Based Pan-Cancer Analysis Highlights Novel Tumor Suppressing Kinases

doi: 10.1101/254813

Figure Lengend Snippet: A) Position of the conserved APE-6 glycine at a hinge point of the activation loop (shown in INSR kinase domain). Active kinase conformation is shown in light blue (PDB ID: 1IR3), inactive kinase conformation is shown in dark blue (PDB ID: 1IRK). DFG and APE motifs are shown as sticks, glycine is shown in sticks and spheres. B) MD simulations show a large decrease in the level of movement observed for the activation loop in MAP2K4 (PDB ID: 3ALN). (i) Root-mean-squared fluctuations (RMSF) of each residue is shown graphically and (ii) structurally, with width and color of the ribbon showing corresponding level of movement. C) Western blot showing biochemically that mutations in the conserved glycine of MAP2K4 are LOF towards to the JNK pathway. A significant decrease in 2D (D) and 3D (matrigel 3D embedded growth assay); E) colony forming potential is observed in CAL51 cell line harboring MAP2K4 G265D following tetracycline-inducible expression of wild type MAP2K4 (*** = p < 0.001). Mutations of the conserved APE-7 glycine within MAP3K13 (F) and PKC6(G) are also LOF. Error bars depict the standard error of the mean; statistical significance was calculated with a two-tailed Students t-test.

Article Snippet: PRKCQ was bought in the pENTR vector (Ultimate Human ORF Library ‐Life Technologies), MAP2K4 and MAP2K7 were bought in pCMV6-Entry vectors (Origene), and MLK4 was purchased in pReceiver-M12 FLAG vector (GeneCopoeia).

Techniques: Activation Assay, Western Blot, Growth Assay, Expressing, Two Tailed Test

Journal: Contrast Media & Molecular Imaging

Article Title: Lidocaine Ameliorates Diabetic Peripheral Neuropathy in Streptozotocin-Induced Diabetic Rats through Modulating the c-Jun Signaling Pathway

doi: 10.1155/2022/1888153

Figure Lengend Snippet: Primer sequences.

Article Snippet: After enclosing with 5% skimmed milk, the antibodies including anti-TNF- α (bs-10802R, 1 : 2, 000, Bioss, China), anti-IL-6 (bs-4539R, 1 : 2, 000, Bioss, China), anti-MKK4 (bs-1977R, 1 : 2, 000, Bioss, China), anti-p-MKK4 (bs-3392R, 1 : 2, 000, Bioss, China), anti-p-JNK (bsm-52462R, 1 : 2, 000, Bioss, China), anti-p-c-Jun (bs-12913R, 1 : 2, 000, Bioss, China), and anti-GAPDH (bs-0755R, 1 : 2, 000, Bioss, China) are incubated overnight.

Techniques:

Journal: eLife

Article Title: Registered report: Diverse somatic mutation patterns and pathway alterations in human cancers

doi: 10.7554/eLife.11566

Figure Lengend Snippet:

Article Snippet: MAP2K4 WT Myc-DDK tagged –includes FLAG tag 1 , Plasmid , Origene , RC206051 , Original product number not specified.

Techniques: Plasmid Preparation, FLAG-tag, Variant Assay, Mutagenesis