Figure S4.
Optimisation of detyrosination activity of vasohibins and CPA enzymes.(A) HeLa cells transduced with lentivirus encoding different constructs of mVash-mSvbp, namely GFP-2A-mSVBP, mVash1-GFP-2A-mSVBP, mVash2_X1-GFP-2A-mSVBP, mVash2_X3-GFP-2A-mSVBP, and GFP-alone were fixed and stained with tyrosination antibody (shown in red color). GFP expression and nuclear staining (DAPI) were shown in gray and blue colors, respectively. Of all the vasohibin-constructs, mVash1 and mVash2_X1 show a clear reduction in microtubule tyrosination levels. Scale bars = 20 µm. (B) HeLa cells transduced with lentivirus encoding different constructs of mVash-mSvbp; GFP-2A-mSVBP, mVash1-GFP-2A-mSVBP, mVash2_X1-GFP-2A-mSVBP, mVash2_X3-GFP-2A-mSVBP, and GFP-alone were fixed and stained with antibodies against tubulin (shown in red color) and detyrosination (shown in green). GFP expression and nuclear staining (DAPI) are shown in gray and blue, respectively. Note that in contrast to mVash1 and mVash2_X3, most of the microtubules in cells expressing mVash2_X1 are detyrosinated. Scale bars = 20 µm. (C) Sequence alignment of mVash1 and splice isoforms of mVash2 (mVash2_X1 and mVash2_X3) is shown. Identical amino acids across these proteins are highlighted in black and amino acids with similar charge are highlighted in gray. mVash2_X1 contains an extra stretch of amino acid sequence in the N-terminal region. (D) Immunoblot analyses of HeLa cell lysates transduced with lentivirus encoding different constructs of mVash-mSVBP. Blots were probed with antibodies against detyrosination, tyrosination, and α-tubulin and with anti-GFP for visualizing the expression of GFP-tagged mVash proteins. Note the increase of detyrosination and decrease of tyrosination in the extracts expressing mVash1 and mVash2_X1. (E) Western blot analysis and quantification of detyrosination levels of CPA-treated tubulin used in Fig. 2 A. The gap lane between CPA-treated and untreated goat brain tubulin lanes showing tubulin staining with antibodies is likely due to spillage from either of the neighboring lanes. A.U., arbitrary units.

Optimisation of detyrosination activity of vasohibins and CPA enzymes. (A) HeLa cells transduced with lentivirus encoding different constructs of mVash-mSvbp, namely GFP-2A-mSVBP, mVash1-GFP-2A-mSVBP, mVash2_X1-GFP-2A-mSVBP, mVash2_X3-GFP-2A-mSVBP, and GFP-alone were fixed and stained with tyrosination antibody (shown in red color). GFP expression and nuclear staining (DAPI) were shown in gray and blue colors, respectively. Of all the vasohibin-constructs, mVash1 and mVash2_X1 show a clear reduction in microtubule tyrosination levels. Scale bars = 20 µm. (B) HeLa cells transduced with lentivirus encoding different constructs of mVash-mSvbp; GFP-2A-mSVBP, mVash1-GFP-2A-mSVBP, mVash2_X1-GFP-2A-mSVBP, mVash2_X3-GFP-2A-mSVBP, and GFP-alone were fixed and stained with antibodies against tubulin (shown in red color) and detyrosination (shown in green). GFP expression and nuclear staining (DAPI) are shown in gray and blue, respectively. Note that in contrast to mVash1 and mVash2_X3, most of the microtubules in cells expressing mVash2_X1 are detyrosinated. Scale bars = 20 µm. (C) Sequence alignment of mVash1 and splice isoforms of mVash2 (mVash2_X1 and mVash2_X3) is shown. Identical amino acids across these proteins are highlighted in black and amino acids with similar charge are highlighted in gray. mVash2_X1 contains an extra stretch of amino acid sequence in the N-terminal region. (D) Immunoblot analyses of HeLa cell lysates transduced with lentivirus encoding different constructs of mVash-mSVBP. Blots were probed with antibodies against detyrosination, tyrosination, and α-tubulin and with anti-GFP for visualizing the expression of GFP-tagged mVash proteins. Note the increase of detyrosination and decrease of tyrosination in the extracts expressing mVash1 and mVash2_X1. (E) Western blot analysis and quantification of detyrosination levels of CPA-treated tubulin used in Fig. 2 A. The gap lane between CPA-treated and untreated goat brain tubulin lanes showing tubulin staining with antibodies is likely due to spillage from either of the neighboring lanes. A.U., arbitrary units.

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