Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Neutrophil Necroptosis Is Triggered by Ligation of Adhesion Molecules following GM-CSF Priming.

doi: 10.4049/jimmunol.1600051

Figure Lengend Snippet: FIGURE 3. PI3Ks are activated by p38 down- stream of adhesion receptors. GM-CSF-primed neutrophils were cultured in the presence and ab- sence of the p38 inhibitor PD169316 (10 mM) or the PI3K inhibitor wortmannin (100 nM), and subsequently stimulated with anti-CD44, anti- CD11b, anti-CD18, or anti-CD15 mAbs for 15 min. Cell lysates were analyzed by immunoblot- ting for phospho-Ser473 AKT, phospho-Thr180/ Tyr182 p38, and phospho-Thr334 MK2. The p38, AKT, and GAPDH expression levels were ana- lyzed as loading controls. Representative immu- noblots are shown (n $ 3).

Article Snippet: Rabbit anti-AKT, rabbit anti-phospho-Ser473-AKT, rabbit anti–phospho-Thr334-MK2 (pMK2), rabbit anti-p38, rabbit anti–phosphoThr180/Tyr182-p38, and rabbit anti-RIPK3 Abs were all from Cell Signaling Technology (Danvers, MA).

Techniques: Cell Culture, Western Blot, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Neutrophil Necroptosis Is Triggered by Ligation of Adhesion Molecules following GM-CSF Priming.

doi: 10.4049/jimmunol.1600051

Figure Lengend Snippet: FIGURE 6. The RIPK3-MLKL pathway is required for p38 activation following ligation of the adhesion molecules CD44, CD11b, CD18, or CD15 in GM-CSF-primed neutro- phils. (A) GM-CSF-primed neutrophils were cultured in the presence and absence of the RIPK1 inhibitor Nec-1 (25 mM), or the RIPK3 inhibitors GSK’843 (50 mM) and GSK’872 (10 mM), and subsequently stimulated with anti-CD44, anti-CD11b, anti-CD18, or anti- CD15 mAbs for 15 min. Cell lysates were analyzed by immunoblotting for phospho- Thr180/Tyr182 p38 and phospho-Thr334 MK2. The p38 and GAPDH expression levels were analyzed as loading controls. (B) The same experiments as described in (A) were performed with the MLKL inhibitor GW806742X (5 mM). (C) Cell lysates of freshly isolated neutrophils from different do- nors were analyzed by immunoblotting for RIPK1, RIPK3, and MLKL. Jurkat cells were analyzed to compare protein levels. GAPDH expression levels were analyzed as load- ing controls. Representative immunoblots are shown (n $ 3).

Article Snippet: Rabbit anti-AKT, rabbit anti-phospho-Ser473-AKT, rabbit anti–phospho-Thr334-MK2 (pMK2), rabbit anti-p38, rabbit anti–phosphoThr180/Tyr182-p38, and rabbit anti-RIPK3 Abs were all from Cell Signaling Technology (Danvers, MA).

Techniques: Activation Assay, Ligation, Cell Culture, Western Blot, Expressing, Isolation

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

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 . Expression of murine MK2 5′UTR isoforms in HeLa cells through introduction of point mutations. After cloning the entire 5′UTR of murine MK2 into peGFP-C1 based vector, different point mutations were introduced into the 5′UTR (see ). HeLa cells were transfected with these plasmids and lysates were analyzed by Western Blot with an anti-MK2 antibody and an anti-p150 antibody to control equal loading. Arrows indicate for the different MK2 isoforms resulting from point mutations within the 5′UTR. B . RAW264.7 and HeLa cells were transfected with an siRNA targeting eIF4A1. Lysates were then analyzed by Western Blot against eIF4A1 to monitor knockdown efficacy and MK2 to detect the endogenous levels of MK2. GAPDH as loading control was used to normalize the signals for quantification (see graph next Western Blot panels). The * indicated for the eIF4A1 band. C . Translation of murine MK2 isoforms upon deletion GC-rich sequences upstream of the CUG aTIS in HeLa and U2OS cells. HeLa and U2OS were either mock-transfected or transfected with the constructs represented next to the Western Blot panels. Lysates were analyzed by Western Blot using an anti-MK2 antibody and an anto-p150 as a loading control. The * indicates for the weak band of the short endogenous human MK2 isoform. D . Schematic representation of the factors that can influence MK2 translation. We confirmed the need of eIF4A1 to promote translation from the CUG aTIS in the 5′UTR of MK2. Adenosine Methylation near the stop codon, as shown indirectly by the knockdown of the methyltransferase Mettl3, and depletion of eIF2A had no effect on MK2 translation in our hands.

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: Expressing, Clone Assay, Plasmid Preparation, Transfection, Western Blot, Construct, Methylation

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 . Quantifications of band intensities from using ImageJ. MK2 protein level were calculated in relation to GAPDH protein level. The line at 0.5 determines the approximate half-life of the MK2 isoforms in both genetic backgrounds. B . Schematic models of the MK2 truncation constructs used to analyze 5′UTR-dependent MK2 translation in . C . Endogenous MK2 isoform translation upon eIF2A knockdown in RAW264.7, wildtype MEFs, HeLa and HEK293T cells. Cells were siRNA transfected with two different specific siRNAs or a control siRNA and lysates were subsequently analyzed by Western Blot for eIF2A to analyze knockdown efficiency and for MK2 to detect translation of isoforms. p150 served as loading control. D . Endogenous MK2 isoform translation upon Mettl3 knockdown in RAW264.7, wildtype MEFs, HeLa and HEK293T cells. Cells were siRNA transfected with two different Mettl3-specific siRNAs or a control siRNA and lysates were subsequently analyzed by Western Blot for Mettl3 to analyze knockdown efficiency and for MK2 to detect translation of isoforms. Tubulin 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: Construct, Transfection, Western Blot

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

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 . Expression of murine MK2 5′UTR isoforms in HeLa cells through introduction of point mutations. After cloning the entire 5′UTR of murine MK2 into peGFP-C1 based vector, different point mutations were introduced into the 5′UTR (see ). HeLa cells were transfected with these plasmids and lysates were analyzed by Western Blot with an anti-MK2 antibody and an anti-p150 antibody to control equal loading. Arrows indicate for the different MK2 isoforms resulting from point mutations within the 5′UTR. B . RAW264.7 and HeLa cells were transfected with an siRNA targeting eIF4A1. Lysates were then analyzed by Western Blot against eIF4A1 to monitor knockdown efficacy and MK2 to detect the endogenous levels of MK2. GAPDH as loading control was used to normalize the signals for quantification (see graph next Western Blot panels). The * indicated for the eIF4A1 band. C . Translation of murine MK2 isoforms upon deletion GC-rich sequences upstream of the CUG aTIS in HeLa and U2OS cells. HeLa and U2OS were either mock-transfected or transfected with the constructs represented next to the Western Blot panels. Lysates were analyzed by Western Blot using an anti-MK2 antibody and an anto-p150 as a loading control. The * indicates for the weak band of the short endogenous human MK2 isoform. D . Schematic representation of the factors that can influence MK2 translation. We confirmed the need of eIF4A1 to promote translation from the CUG aTIS in the 5′UTR of MK2. Adenosine Methylation near the stop codon, as shown indirectly by the knockdown of the methyltransferase Mettl3, and depletion of eIF2A had no effect on MK2 translation in our hands.

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: Expressing, Clone Assay, Plasmid Preparation, Transfection, Western Blot, Construct, Methylation

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 . Quantifications of band intensities from using ImageJ. MK2 protein level were calculated in relation to GAPDH protein level. The line at 0.5 determines the approximate half-life of the MK2 isoforms in both genetic backgrounds. B . Schematic models of the MK2 truncation constructs used to analyze 5′UTR-dependent MK2 translation in . C . Endogenous MK2 isoform translation upon eIF2A knockdown in RAW264.7, wildtype MEFs, HeLa and HEK293T cells. Cells were siRNA transfected with two different specific siRNAs or a control siRNA and lysates were subsequently analyzed by Western Blot for eIF2A to analyze knockdown efficiency and for MK2 to detect translation of isoforms. p150 served as loading control. D . Endogenous MK2 isoform translation upon Mettl3 knockdown in RAW264.7, wildtype MEFs, HeLa and HEK293T cells. Cells were siRNA transfected with two different Mettl3-specific siRNAs or a control siRNA and lysates were subsequently analyzed by Western Blot for Mettl3 to analyze knockdown efficiency and for MK2 to detect translation of isoforms. Tubulin 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: Construct, Transfection, Western Blot

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 . Expression profiling of MK2 in different murine and human cell lines. Lysates from the indicated cell lines were subjected to Western Blot analysis using an anti-MK2 antibody (D1E11, Cell Signaling) and an anti-GAPDH as loading control. B . Expression of MK2 in several mouse organs as detected by Western Blot analyses using a MK2-specific and a MK3-specific antibody. The GAPDH development served as a loading control. C . Genomic tracks of RNASeq and RiboSeq data sets (GSE81250) for the old murine MK2 transcripts NM_008551.1 and its newest annotated transcript version NM_008551.2. The position of potential alternative translation initiation sites (aTIS) within the 5′UTR of MK2 and the AUG initiation sites are indicated. The length of both 5′UTR are given (18 and 318 nucleotides, respectively). D . The murine MK2 5′UTR nucleotide sequence and its putative extended in-frame N-terminal amino acid sequence. The aTIS are marked in bold and in italics within both nucleotide and the amino acid sequence. E . Purification of endogenous MK2 isoforms form RAW264.7 cells. Endogenous MK2 was purified from 1 × 10 8 RAW264.7 cells using an agarose MK2-Trap (Chromotek). Input (lysate), column flow-through and precipitates (beads, eluted using 2x SDS loading buffer) were separated on a 10% SDS gel that was Coomassie blue stained afterwards. Bands were excised and further processed for mass-spectrometry analysis. F . Identification of MK2 peptides from mass-spectrometry (1E.). The three translation initiation sites are marked in bold and italics. Red sequences depict detected peptides and black ones the non-detected.

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: Expressing, Western Blot, Sequencing, Purification, SDS-Gel, Staining, Mass Spectrometry

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 . Genomic tracks for MK2 transcripts NM_004759.4 and NM_032960.3 in Ribosome Footprinting analyses derived from two different HEK293 data sets (GSE70804 and GSE37744). B . Human MK2 5′UTR nucleotide sequence and its putative extended in-frame N-terminal amino acid sequence. The aTIS are marked in bold and in italics within both nucleotide and the amino acid sequence. C . Alignment of the murine and human MK2 5′UTR nucleotide (lower) and amino acid (upper panel) sequences. The positions of putative aTIS are marked by red arrows within the amino acid sequence alignments.

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: Footprinting, Derivative Assay, Sequencing

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: Upper panel (GSE74139): BMDCs were treated with cycloheximide (CHX), Lactimidomycin (LTM) or Harringtonine (Har) to freeze ribosomes on the mRNAs at different positions. Lower panel (GSE83332): Keatinocytes were either treated with cycloheximide (CHX) or Harringtonine (Har) to freeze ribosome on mRNAs in order to detect their positions form sequencing results. In both analyses the position of ribosomal footprints is shown for the 5′UTR of the murine MK2 transcript NM_008551.2.

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: Sequencing

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 . Analysis of TTP, cFos and Egr1 expression upon Anisomycin stimulation for the indicated times in differentially rescued MK2-deficient MEFs. Each data point represents a biological replicate that was measured in duplicates. Error bars represent the mean of three independent experiments (n = 3) +/ − the standard deviation. P-values were retrieved from one-way ANOVA testing using the PRISM 7 software and are related to GFP/EV-rescued MEFs. B . Fluorescence-based scratch wound healing assay for differentially rescued MK2-deficient MEFs as shown before . Each individual data point represents a biological replicate (n > 18) of the analysis and the mean of all replicates is shown with its standard deviations. P-values were retrieved from one-way ANOVA testing using the PRISM 7 software and are related to GFP/EV-transduced MK2-deficient MEFs. C . The interaction of MK2 isoforms with their endogenous upstream activating kinase p38 MAPK was detected by co-immunoprecipitation experiments. Non-stimulated and Anisomycin-stimulated MK2/3 DKO complemented MEFs were analyzed. A MK2-Trap was used to precipitate MK2 effectively. Beads were eluted using 2x SDS loading buffer and samples were analyzed by Western Blot development for MK2 and p38 MAPK . eEF2 development served as a control. D . Volcano plots of significantly MK2 short and MK2 long interacting proteins at the indicated conditions. Selected proteins are highlighted in red. The black lines define the border of significantly and non-significantly interacting proteins.

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: Expressing, Standard Deviation, Software, Fluorescence, Wound Healing Assay, Immunoprecipitation, Western Blot

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 . Schematic representation of the MK2 5′UTR cloning strategy. The hitherto known murine MK2 coding sequence was cloned into peGFP-C1 allowing its expression in mammalian cells with a N-terminal GFP-fusion. By cutting with NheI (right after the CMV promotor of peGFP-C1) and PstI (within the known MK2 coding sequence), this part was replaced with a synthesized 5′UTR sequence containing the same sites at the 3’ and 5’ ends. This resulted in the elimination of the GFP-tag, but CMV-promotor driven expression of the complete 5′UTR in fusion with the MK2 coding sequence. This construct then contained the natural AUG translation initiation site and the other two potential aTIS CUG and GUG in position –141 and –309, respectively. The table below shows the different mutants that were derived from the initial construct #1 that contained the wildtype 5′UTR sequence. The predicted consequences of the mutations are given in the last column of the table. B . Expression of MK2 mutants in U2OS cells after transient transfection. The same plasmids as in were transfected into U2OS cells to prove effect the predicted consequences from the given in S5A. Lysates of transfected cells were analyzed by Western Blot with a MK2-specific antibody. Loading control: anti-p150.

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: Clone Assay, Sequencing, Expressing, Synthesized, Construct, Derivative Assay, Transfection, Western Blot

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 . Expression of murine MK2 5′UTR isoforms in HeLa cells through introduction of point mutations. After cloning the entire 5′UTR of murine MK2 into peGFP-C1 based vector, different point mutations were introduced into the 5′UTR (see ). HeLa cells were transfected with these plasmids and lysates were analyzed by Western Blot with an anti-MK2 antibody and an anti-p150 antibody to control equal loading. Arrows indicate for the different MK2 isoforms resulting from point mutations within the 5′UTR. B . RAW264.7 and HeLa cells were transfected with an siRNA targeting eIF4A1. Lysates were then analyzed by Western Blot against eIF4A1 to monitor knockdown efficacy and MK2 to detect the endogenous levels of MK2. GAPDH as loading control was used to normalize the signals for quantification (see graph next Western Blot panels). The * indicated for the eIF4A1 band. C . Translation of murine MK2 isoforms upon deletion GC-rich sequences upstream of the CUG aTIS in HeLa and U2OS cells. HeLa and U2OS were either mock-transfected or transfected with the constructs represented next to the Western Blot panels. Lysates were analyzed by Western Blot using an anti-MK2 antibody and an anto-p150 as a loading control. The * indicates for the weak band of the short endogenous human MK2 isoform. D . Schematic representation of the factors that can influence MK2 translation. We confirmed the need of eIF4A1 to promote translation from the CUG aTIS in the 5′UTR of MK2. Adenosine Methylation near the stop codon, as shown indirectly by the knockdown of the methyltransferase Mettl3, and depletion of eIF2A had no effect on MK2 translation in our hands.

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: Expressing, Clone Assay, Plasmid Preparation, Transfection, Western Blot, Construct, Methylation

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 . Quantifications of band intensities from using ImageJ. MK2 protein level were calculated in relation to GAPDH protein level. The line at 0.5 determines the approximate half-life of the MK2 isoforms in both genetic backgrounds. B . Schematic models of the MK2 truncation constructs used to analyze 5′UTR-dependent MK2 translation in . C . Endogenous MK2 isoform translation upon eIF2A knockdown in RAW264.7, wildtype MEFs, HeLa and HEK293T cells. Cells were siRNA transfected with two different specific siRNAs or a control siRNA and lysates were subsequently analyzed by Western Blot for eIF2A to analyze knockdown efficiency and for MK2 to detect translation of isoforms. p150 served as loading control. D . Endogenous MK2 isoform translation upon Mettl3 knockdown in RAW264.7, wildtype MEFs, HeLa and HEK293T cells. Cells were siRNA transfected with two different Mettl3-specific siRNAs or a control siRNA and lysates were subsequently analyzed by Western Blot for Mettl3 to analyze knockdown efficiency and for MK2 to detect translation of isoforms. Tubulin 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: Construct, Transfection, Western Blot

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

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 . Determining the growth of complemented MK2/3 DKO MEFs by a WST-1 assays. 1 × 10 4 cells of every genotype were seeded in triplicates for each timepoint in a 96-well plate format. At the indicated times WST-1 was added to the cells and after 30 minutes of incubation at 37°C, densiometric measurements were performed in a multi-well plate reader. Relative OD600nm are plotted against the time [days]. B . Rescued MK2 MEFs were starved for 24h in serum-free DMEM and afterwards stimulated with 10% FCS-containing DMEM for 1 and 2h. Lysates were consequently analyzed for the expression of the indicated proteins. C . Expression of TTP mRNA in rescued MK2 MEFs after starvation as in Fig. S9B and stimulation with 10% FCS-containing DMEM for 0, 2 and 4h. Error bars represent the mean of three independent experiments (n = 3) +/− the standard deviation. P-values were retrieved from one-way ANOVA testing using the PRISM 7 software and are related to GFP/EV-rescued MEFs.

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: Incubation, Expressing, Standard Deviation, Software

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