<i>mpk-1</i> Expression in the C. elegans Germline

<div><p>(A) Schematics of <i>mpk-1a</i> and <i>mpk-1b</i> mRNAs. Box, exon; connecting line, intron; ATG, initiation codon; TAG, termination codon. Below schematics: thick bars, extent of probes used for in situ hybridization; arrows, primer pairs used for RT-PCR.</p><p>(B) Semiquantitative RT-PCR of RNA prepared from adult hermaphrodites that either had an essentially normal germline [<i>glp-1(q224)</i> grown at 15 °C], or had virtually no germline [<i>glp-1(q224)</i> grown at 25 °C] (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030233#s4" target="_blank">Materials and Methods</a>). <i>unc-54</i> was used as a control.</p><p>(C) Western blot. MPK-1A protein is ≈45 kDa, MPK-1B is ≈55 kDa, and α-TUB is α-tubulin. Proteins were extracted from adult hermaphrodites that were either wild-type (wt), <i>glp-1(q224)</i> grown at 25 °C (GL−), or <i>mpk-1(ga117)</i> putative null homozygotes <i>[mpk-1(0)]</i>.</p><p>(D–F) In situ analysis of dissected adult hermaphrodite germlines. (D) Total <i>mpk-1</i> RNA was assessed using the <i>mpk-1ab</i> antisense probe shown in (A). (E) <i>mpk-1b</i> RNA was assessed using an isoform-specific antisense probe shown in (A). (F) Negative control, using an <i>mpk-1b</i>−specific sense probe.</p><p>(G–L) Immunocytochemistry of dissected adult hermaphrodite germlines. All were stained using both MPK-1 antibodies (G, I, K) and DAPI (H, J, L). Distal end, arrowhead; dotted lines, boundaries between regions of germline maturation [MR (mitotic region), TZ (transition zone), PR (pachytene region), OO (oocytes), SP (sperm)]; PEX (pachytene exit defect). (G, H) Same wild-type germline. (I, J) Same <i>mpk-1(0)</i> germline. (K, L) Same <i>mpk-1b(RNAi)</i> germline.</p>