Journal: The Journal of Biological Chemistry
Article Title: Biological Significance of Nuclear Localization of Mitogen-activated Protein Kinase Pmk1 in Fission Yeast
Figure Lengend Snippet: Cell wall integrity is partially compromised in cells expressing a nuclear-excluded version of Pmk1. A, strains LS111 (Pmk1-GFP; control), MI102 (pmk1Δ), LS115 (Pmk1-GFP-CAAX), LS137 (Pmp1-GFP-RitC), LS127 (mbx1Δ, Pmk1-GFP), and LS128 (mbx1Δ, Pmk1-GFP-CAAX) were grown in YES medium (A600 = 0.5) and assayed for β-glucanase sensitivity. Cell lysis was monitored by measuring decay in A600 for different incubation periods. Results shown are the mean value of three independent experiments. B, cell wall composition of strains LS111 (control), MI102, and LS115 is shown. Strains were grown in YES medium at 32 °C, and [14C]glucose was added 6 h before harvesting. Values shown are the mean of three independent experiments with duplicate samples, and error bars represent S.D. for total carbohydrate values. C, caspofungin sensitivity assays for strains LS111 (Pmk1-GFP; control), MI102 (pmk1Δ), LS115 (Pmk1-GFP-CAAX), LS116 (Pmk1(K52E)-GFP-CAAX; kinase dead), and LS137 (Pmk1-GFP-RitC) are shown. After growth in YES medium, 104, 103, 102, or 10 cells were spotted onto YES plates supplemented with 0.8, 1.0, or 1.2 μg/ml caspofungin and incubated for 3 days at 28 °C before being photographed. D, strains LS111 (Pmk1-GFP; control) and LS115 (Pmk1-GFP-CAAX) were grown in YES medium to mid log-phase and treated with 1 μg/ml caspofungin. At timed intervals either activated or total Pmk1 was detected by immunoblotting with anti-phospho-p42/44 or anti-GFP antibodies, respectively. E, shown are caspofungin sensitivity assays for strains LS111, LS115, LS132 (sty1Δ, Pmk1-GFP), L133 (sty1Δ, Pmk1-GFP-CAAX), MI102, LS108 (mbx1Δ), LS109 (mbx1Δ pmk1Δ), LS127 (mbx1Δ, Pmk1-GFP), LS128 (mbx1Δ, Pmk1-GFP-CAAX), LS106 (atf1Δ), LS107 (atf1Δ pmk1Δ), LS125 (atf1Δ, Pmk1-GFP), LS126 (atf1Δ, Pmk1-GFP-CAAX), LS110 (mbx1Δ atf1Δ pmk1Δ), LS129 (mbx1Δ atf1Δ, Pmk1-GFP), and LS130 (mbx1Δ atf1Δ, Pmk1-GFP-CAAX).
Article Snippet: The lysates were cleared by centrifugation at 13,000 rpm for 15 min, and the proteins were resolved in 10% SDS-PAGE gels, transferred to nitrocellulose filters, and incubated with either polyclonal rabbit anti-GFP (Cell Signaling) or polyclonal rabbit anti-phospho-p42/44 antibodies (Cell Signaling).
Techniques: Expressing, Lysis, Incubation, Western Blot