Journal: Kinetoplastid Biology and Disease

Article Title: LmxMPK4, a mitogen-activated protein (MAP) kinase homologue essential for promastigotes and amastigotes of Leishmania mexicana

doi: 10.1186/1475-9292-4-6

Figure Lengend Snippet: Southern analysis of genomic DNA . 1, Pst I; 2, Hin cII; 3, Hin dIII; 4, Acc 65I; 5, Xba I; 6, Spe I; 7, Eco RV; 8, Cla I; 9, Nde I; 10, Nco I; 11, Mlu I; 12, Mun I. Digested DNA (5 μg) was electrophoresed on a 0.7% agarose gel, blotted onto nylon membranes and probed with a DIG-labelled DNA probe corresponding to a fragment of the open reading frame (nt 240 – nt 930). Numbers indicate the approximate size of DNA markers in kb.

Article Snippet: Gene cloning, sequencing and analysis Positive phage clones for LmxMPK4 were selected using digoxigenin (DIG)-labelled probes [ ], amplified and DNA inserts cloned into pBSKII(+) using Eco RI (Stratagene, La Jolla, CA) yielding pBE25LmxMPK4.

Techniques: Agarose Gel Electrophoresis

Journal: Kinetoplastid Biology and Disease

Article Title: LmxMPK4, a mitogen-activated protein (MAP) kinase homologue essential for promastigotes and amastigotes of Leishmania mexicana

doi: 10.1186/1475-9292-4-6

Figure Lengend Snippet: Deletion analysis of LmxMPK4 . Southern blot analysis of different clones with a probe corresponding to the 3'-UTR of LmxMPK4 . 1, L. mexicana wild type; 2, episome pXPACLmxMPK4 + Δ LmxMPK4 +/- Phleo; 3, episome pXPACLmxMPK4 + Δ LmxMPK4 +/- Hyg; 4, episome pXPACLmxMPK4 + Δ LmxMPK4 -/-/+ Phleo/Hyg; 5, episome pXPACLmxMPK4 + Δ LmxMPK4 -/- Phleo/Hyg. Numbers indicate the approximate size of DNA markers in kb.

Article Snippet: Gene cloning, sequencing and analysis Positive phage clones for LmxMPK4 were selected using digoxigenin (DIG)-labelled probes [ ], amplified and DNA inserts cloned into pBSKII(+) using Eco RI (Stratagene, La Jolla, CA) yielding pBE25LmxMPK4.

Techniques: Southern Blot, Clone Assay

Journal: Kinetoplastid Biology and Disease

Article Title: LmxMPK4, a mitogen-activated protein (MAP) kinase homologue essential for promastigotes and amastigotes of Leishmania mexicana

doi: 10.1186/1475-9292-4-6

Figure Lengend Snippet: Deletion analysis of LmxMPK4 . Southern blot analysis of different clones with a probe corresponding to the 3'-UTR of LmxMPK4 . 1, L. mexicana wild type; 2, episome pXPACLmxMPK4 + Δ LmxMPK4 +/- Phleo; 3, episome pXPACLmxMPK4 + Δ LmxMPK4 +/- Hyg; 4, episome pXPACLmxMPK4 + Δ LmxMPK4 -/-/+ Phleo/Hyg; 5, episome pXPACLmxMPK4 + Δ LmxMPK4 -/- Phleo/Hyg. Numbers indicate the approximate size of DNA markers in kb.

Article Snippet: A rabbit antiserum was produced against the peptide CEDLWRVVEAHSQLNE corresponding to the 15 carboxy-terminal amino acids of LmxMPK4 (Eurogentec, Seraing, Belgium).

Techniques: Southern Blot, Clone Assay

Journal: Kinetoplastid Biology and Disease

Article Title: LmxMPK4, a mitogen-activated protein (MAP) kinase homologue essential for promastigotes and amastigotes of Leishmania mexicana

doi: 10.1186/1475-9292-4-6

Figure Lengend Snippet: Alignment of LmxMPK4 from L. mexicana with various MAP kinase amino acid sequences . LmxMPK4, Leishmania mexicana MAP kinase 4 (GenBank accession number AJ293282 ; [6]); Trypanosoma brucei MAP kinase, TbMAPK2 (GeneDB accession number Tb10.70.2070; [10]); Trypanosoma cruzi MAP kinase (GeneDB accession number Tc00.1047053511299.70); Chlamydomonas reinhardtii MAP kinase (GenBank accession number BAB18271 ); Dictyostelium discoideum ERK1 (GenBank accession number U11077 ; [13]); Homo sapiens ERK2 (GenBank accession number CAA77753 ; [14]). Roman numerals I to XI indicate kinase subdomains. The kinase domain is located between the two inverted triangles (▼). Arrows (↓) mark the potential regulatory phosphorylation sites in the TXY motif at Thr190 and Tyr192, filled circles (●) indicate conserved amino acid residues it is not clear what you mean by conserved aa residues, and open circles (○) depict the residues of the phosphate anchor ribbon for ATP binding. The asterisks (*) show the residues of the P+1 specificity pocket. The open triangle (Δ) marks the invariant lysine residue (K59), which is essential for the phosphate transfer reaction. The CD-domain is shaded in light grey. Dashes indicate gaps introduced to optimize the alignment; dots represent identical residues. The carboxy-terminal peptide used to generate antibodies is underlined. (50), the terminal 50 amino acid residues of the Dictyostelium kinase have been omitted. Numbering corresponds to LmxMPK4.

Article Snippet: A rabbit antiserum was produced against the peptide CEDLWRVVEAHSQLNE corresponding to the 15 carboxy-terminal amino acids of LmxMPK4 (Eurogentec, Seraing, Belgium).

Techniques: Binding Assay

Journal: Kinetoplastid Biology and Disease

Article Title: LmxMPK4, a mitogen-activated protein (MAP) kinase homologue essential for promastigotes and amastigotes of Leishmania mexicana

doi: 10.1186/1475-9292-4-6

Figure Lengend Snippet: Immunoblot of LmxMPK4 from L. mexicana wild type and episomal overexpressor . Lane 1, L. mexicana wild type promastigotes (4 × 10 7 cells); lane 2, amastigotes derived from mouse lesion (4 × 10 7 cells); lane 3, wild type in vitro differentiated amastigotes (1 × 10 8 cells); lane 4, promastigotes of LmxMPK4 episomal overexpressor (1 × 10 7 cells). A, blot probed with antiserum against the carboxy-terminal peptide of LmxMPK4; B, the blot was stripped and re-probed with a polyclonal antiserum against myo-inositol-1-phosphate synthase. The molecular masses of standard proteins are indicated in kilodaltons.

Article Snippet: A rabbit antiserum was produced against the peptide CEDLWRVVEAHSQLNE corresponding to the 15 carboxy-terminal amino acids of LmxMPK4 (Eurogentec, Seraing, Belgium).

Techniques: Derivative Assay, In Vitro

Journal: Kinetoplastid Biology and Disease

Article Title: LmxMPK4, a mitogen-activated protein (MAP) kinase homologue essential for promastigotes and amastigotes of Leishmania mexicana

doi: 10.1186/1475-9292-4-6

Figure Lengend Snippet: Recombinant expression of LmxMPK4 and kinase assay . Left panels, Coomassie-stained gels; right panels, autoradiographs. A, recombinant GST-fusion proteins after kinase assay. Lanes 1 and 1', wild type LmxMPK4; lane 2 and 2', KM-mutant of LmxMPK4. B, kinase assay of GST-LmxMPK4 at different concentrations of Mg 2+ and Mn 2+ . Lanes 1 and 1', 50 mM Mg 2+ ; lanes 2 and 2', 10 mM Mg 2+ ; lanes 3 and 3', 5 mM Mg 2+ ; lanes 4 and 4', 10 mM Mg 2+ and 2 mM Mn 2+ ; lanes 5 and 5', 10 mM Mn 2+ ; lanes 6 and 6', 2 mM Mn 2+ ; lanes 7 and 7', 1 mM Mn 2+ . C, kinase assay of GST-LmxMPK4 at varying pH. Lanes 1 and 1', pH5.5; lanes 2 and 2', pH6.0; lanes 3 and 3', pH6.5; lanes 4 and 4', pH7.0; lanes 5 and 5', pH7.2; lanes 6 and 6', pH7.5; lanes 7 and 7', pH8.0; lanes 8 and 8', pH 8.5. Molecular masses of standard proteins are indicated in kilodalton.

Article Snippet: A rabbit antiserum was produced against the peptide CEDLWRVVEAHSQLNE corresponding to the 15 carboxy-terminal amino acids of LmxMPK4 (Eurogentec, Seraing, Belgium).

Techniques: Recombinant, Expressing, Kinase Assay, Staining, Mutagenesis