anti-pmk2-t334  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Alternative translation initiation generates a functionally distinct isoform of the stress-activated kinase MK2

doi: 10.1101/429696

Figure Lengend Snippet: A . Detection of MK2, its phosporylated form (pMK2 T222 ) and MK3 in MK2 KO and MK2/3 DKO bone marrow-derived macrophages (BMDMs) upon LPS-stimulation. The star indicates for a cross-reactive non-specific band detected by the MK2 antibody. Loading control: GAPDH B . Confirmation of the MK3-specific band in MK2 versus MK2/3 DKO MEFs by analyzing lysates of the indicted cells by Western Blot with a MK3-specific antibody. C . Detection of MK2 isoforms and MK3 in Jurkat-E6 and HCT116 cells by Western Blot using specific anti-MK2 and anit-MK3 antibodies. Loading control: GAPDH D . shRNA-mediated knockdown of MK2 in HT29 cells using different shRNAs raised against human MK2 (Particle #TRCN0000002282 and # TRCN0000002283) as well as a control shRNA (shCtrl) and magnitudes of infection (MOIs) were used. The efficiency of knockdown was analyzed by Western Blot of lysates probed with an MK2 antibody. Anti-GAPDH development served as a loading control.

Article Snippet: Antibodies to detect specific proteins: anti-MK2 (CST, #3042 and #12155), anti-pMK2 T222 (CST, #3316), anti-pMK2 T334 (CST, #3041), anti-MK3 (CST, #3034), anti-p38 (CST, #9012), antipp38 T180/Y182 (CST, #4511), anti-NOGO-B (R+D Systems, AF6034), anti-Hsp27 S82 (CST, #9709), anti-GAPDH (EMD Millipore, #MAB374), anti-Tubulin (Sigma, #T6199), anti-eEF2 (SCBT, sc-13004), anti-p150 (BD Biosciences, #610473), anti-Mettl3 (CST, #96391), antieIF4A1 (CST, #2490T), anti-eIF2A (Proteintech, 11233–1-AP), anti-GFP (SCBT, sc-9996) and anti-TTP (N-Term) (Sigma T5327) HRP-coupled secondary antibodies were used for ECL-based detection of proteins on either a LAS3000 FujiFilm or a Bio-Rad ChemiDoc Imaging System.

Techniques: Derivative Assay, Western Blot, shRNA, Infection

Journal: bioRxiv

Article Title: Alternative translation initiation generates a functionally distinct isoform of the stress-activated kinase MK2

doi: 10.1101/429696

Figure Lengend Snippet: A . Complementation of MK2 KO and MK2/3 DO mouse embryonic fibroblasts (MEFs) by retroviral transduction. Subcloning of 5′UTR mutants into pMMP-MK2-IRES-eGFP based vectors and subsequent viral transduction of MK2-deficient and MK2/3 double-deficient MEFs resulted in expression of either the short or the long MK2 isoform only, both isoforms in parallel (both) or a super-long (super) isoform. Empty vector transduced cells served as controls and non-transduced murine cell lines (RAW264.7, NIH3T3 and wildtype (WT) MEFs) indicated the position of endogenous MK2 isoforms and expression levels in transduced cell lines. B . The stability of MK2 isoforms in MK2 KO or MK2/3 DKO complemented MEFs was analyzed after incubating cells with 20 µg/ml cycloheximide (CHX) for the indicated times. A quantification of the band intensities can be found in . The GAPDH development served as a loading control. C . The differentially complemented MK2/3 DKO MEFs from were analyzed for the phosphorylation of known MK2 substrates (NOGO-B and Hsp27) upon Anisomycin stimulation. Phosphorylation of the MK2 upstream p38 MAPK (pp38 T180/Y182 ) and MK2 (pMK2 T 334) itself was used as a readout for activation of the pathway. To control activity, cells were either pre-incubated with an p38 MAPK inhibitor (Birb796) or a MK2 inhibitor (PF-3644022). p150 development served as loading control.

Article Snippet: Antibodies to detect specific proteins: anti-MK2 (CST, #3042 and #12155), anti-pMK2 T222 (CST, #3316), anti-pMK2 T334 (CST, #3041), anti-MK3 (CST, #3034), anti-p38 (CST, #9012), antipp38 T180/Y182 (CST, #4511), anti-NOGO-B (R+D Systems, AF6034), anti-Hsp27 S82 (CST, #9709), anti-GAPDH (EMD Millipore, #MAB374), anti-Tubulin (Sigma, #T6199), anti-eEF2 (SCBT, sc-13004), anti-p150 (BD Biosciences, #610473), anti-Mettl3 (CST, #96391), antieIF4A1 (CST, #2490T), anti-eIF2A (Proteintech, 11233–1-AP), anti-GFP (SCBT, sc-9996) and anti-TTP (N-Term) (Sigma T5327) HRP-coupled secondary antibodies were used for ECL-based detection of proteins on either a LAS3000 FujiFilm or a Bio-Rad ChemiDoc Imaging System.

Techniques: Transduction, Subcloning, Expressing, Plasmid Preparation, Activation Assay, Activity Assay, Incubation

Journal: bioRxiv

Article Title: Alternative translation initiation generates a functionally distinct isoform of the stress-activated kinase MK2

doi: 10.1101/429696

Figure Lengend Snippet: A . Transfected HeLa cells were analyzed for the expression GFP-tagged MK2 isoforms. The initial N-terminal tagged GFP-MK2 construct (peGFP-C1-MK2, see Fig. S5A) was used as a control as well as the peGFP-N1 vector used to clone the C-terminally tagged construct used in fluorescence microscopy. Western Blots were developed with anti-MK2- and anti-GFP-specific antibodies. Loading control: GAPDH. The translocation of MK2 isoforms from the nucleus to the cytosol after exposition to Anisomycin for 60 minutes was analyzed by fluorescence microscopy. U2OS cells were transfected with plasmids coding for C-terminal tagged isoforms of murine MK2. For this the mutant (construct #3 and #4, see and ) were subcloned into peGFP-N1 vectors that allow C-terminal GFP-tagging of inserted coding sequences. B . MEFs were analyzed for the phosphorylation of known MK2 substrates (NOGO-B and Hsp27) upon Anisomycin stimulation. Phosphorylation of the MK2 upstream p38 MAPK (pp38 T180/Y182 ) and MK2 (pMK2 T334 ) itself was used as a readout for pathway activation. To control activity, cells were either pre-incubated with an p38 MAPK inhibitor (Birb796) or a MK2 inhibitor (PF-3644022). p150 development served as loading control.

Article Snippet: Antibodies to detect specific proteins: anti-MK2 (CST, #3042 and #12155), anti-pMK2 T222 (CST, #3316), anti-pMK2 T334 (CST, #3041), anti-MK3 (CST, #3034), anti-p38 (CST, #9012), antipp38 T180/Y182 (CST, #4511), anti-NOGO-B (R+D Systems, AF6034), anti-Hsp27 S82 (CST, #9709), anti-GAPDH (EMD Millipore, #MAB374), anti-Tubulin (Sigma, #T6199), anti-eEF2 (SCBT, sc-13004), anti-p150 (BD Biosciences, #610473), anti-Mettl3 (CST, #96391), antieIF4A1 (CST, #2490T), anti-eIF2A (Proteintech, 11233–1-AP), anti-GFP (SCBT, sc-9996) and anti-TTP (N-Term) (Sigma T5327) HRP-coupled secondary antibodies were used for ECL-based detection of proteins on either a LAS3000 FujiFilm or a Bio-Rad ChemiDoc Imaging System.

Techniques: Transfection, Expressing, Construct, Plasmid Preparation, Fluorescence, Microscopy, Western Blot, Translocation Assay, Mutagenesis, Activation Assay, Activity Assay, Incubation