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 . 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