Journal: bioRxiv

Article Title: TFIIB-related factor 1 is a nucleolar protein that promotes RNA polymerase I-directed transcription and tumour cell growth

doi: 10.1101/2022.02.20.481212

Figure Lengend Snippet: (A) BRF1 and TIF-1A were co-localized in the nucleoli of HeLa cells. IF staining was performed using the BRF1 and TIF-1A antibodies. The scale bar in each image represents 2.5 μm. ( B ) BRF1 and RPA40 were co-localized in the nucleoli of HeLa cells. IF staining was performed using BRF1 antibody and RPA40 antibody. The scale bar in each image represents 2.5 μm. ( C ) The co-localization analysis between BRF1 and TIF-1A using the nucleolar particles purified from HeLa cells. The scale bar in each image represents 2.5 μm. ( D ) The co-localization analysis between BRF1 and RPA43 using the nucleoli particles purified from HeLa cells. The scale bar in each image represents 5 μm. ( E ) Components of the Pol I transcription machinery could be precipitated by the BRF1 antibody. HeLa nuclear extract was prepared and used for BRF1 IP assays. BRF1-binding proteins in the IP samples were detected by Western blot using the antibodies as indicated in the panel. ( F , G ) BRF1 could be precipitated by the antibodies against Pol I-related proteins. IP assays were performed with the antibodies against TBP, TIF-1A, TAF1A, UBF, and RPA43, respectively. BRF1 ( F ) and proteins as indicated ( G ) were detected by Western blot. Figure 6-figure supplement 1. The co-localization analysis between BRF1 and Pol III transcription factors in HeLa cells. Figure 6-figure supplement 2. BRF1 binds to TFIIIC subunit GTF3C2 and SL1 subunit TAF1B. Figure 6-source data, Figure 6-figure supplement 2-source data

Article Snippet: IP assays were performed using normal IgG, BRF1 antibody and the antibodies against UBF (ab244287, Abcam), TIF-1A (ab251933, Abcam), TBP (ab28175, abcam), TAF1A (SC-393600, Santa Cruz Biotech), GTF3C2 (SC-81406, Santa Cruz Biotech), TAF1B (CSB-PA684476ESR1HU, CUSABio) and RPA43 (Ab99305, Abcam), respectively.

Techniques: Staining, Purification, Binding Assay, Western Blot

Journal: bioRxiv

Article Title: TFIIB-related factor 1 is a nucleolar protein that promotes RNA polymerase I-directed transcription and tumour cell growth

doi: 10.1101/2022.02.20.481212

Figure Lengend Snippet: (A) BRF1 IP results showing BRF1 association with GTF3C2 and TAF1B. IP assays were performed using BRF1 antibody and HeLa nuclear extract. BRF1-bound protein was detected by Western blot using the antibodies as indicated. ( B ) GTF3C2 IP result showing GT3C2 association with BRF1. IP assays were performed using GTF3C2 antibody and HeLa nuclear extract. GTF3C2-bound protein was detected by Western blot using the antibodies as indicated. ( C ) TAF1B IP result showing TAF1B association with BRF1. IP assays were performed using TAF1B antibody and HeLa nuclear extract. TAF1B-bound protein was detected by Western blot using the antibodies as indicated.

Article Snippet: IP assays were performed using normal IgG, BRF1 antibody and the antibodies against UBF (ab244287, Abcam), TIF-1A (ab251933, Abcam), TBP (ab28175, abcam), TAF1A (SC-393600, Santa Cruz Biotech), GTF3C2 (SC-81406, Santa Cruz Biotech), TAF1B (CSB-PA684476ESR1HU, CUSABio) and RPA43 (Ab99305, Abcam), respectively.

Techniques: Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Biological Significance of Nuclear Localization of Mitogen-activated Protein Kinase Pmk1 in Fission Yeast

doi: 10.1074/jbc.M112.345611

Figure Lengend Snippet: Effect of nuclear exclusion of Pmk1 on chloride homeostasis. A, upper panel, cells from strains LS111 (Pmk1-GFP) (control), LS115 (Pmk1-GFP-CAAX), LS119 (rho2Δ, Pmk1-GFP), LS120 (rho2Δ, Pmk1-GFP-CAAX), LS121 (pck2Δ, Pmk1-GFP), and LS122 (pck2Δ, Pmk1-GFP-CAAX) were grown in YES medium to mid-log phase. Basal Pmk1 activity was detected by immunoblotting of cell extracts with anti-(phosho-p42/44) antibody, and total Pmk1 was detected with anti-GFP antibody as loading control (R.U. indicates relative units). Lower panel, cells from strains LS111 (Pmk1-GFP) (control), LS115 (Pmk1-GFP-CAAX), and LS137 (Pmk1-GFP-RitC) were grown in YES medium, and basal Pmk1 activity was detected and quantified as described above. B, shown are vic phenotype and chloride sensitivity assays for strains LS111 (control), MI102 (pmk1Δ), LS115, and LS116 (Pmk1(K52E)-GFP-CAAX; kinase dead). After growth in YES medium, 104, 103, 102, or 10 cells were spotted onto YES plates supplemented with 0.2 m MgCl2 plus 0.5 μg/ml FK506 (vic) or with 0.2–0.3 M MgCl2 alone and incubated for 3 days at 28 °C before being photographed. C, basal Pmk1 activity in growing cells from strains LS111 (control), LS115, LS117 (pmp1Δ, Pmk1-GFP), LS118 (pmp1Δ, Pmk1-GFP-CAAX), LS132 (sty1Δ, Pmk1-GFP), and LS133 (sty1Δ, Pmk1-GFP-CAAX) was detected as described above. D, chloride sensitivity assay for the strains described in C is shown. E, strains LS138 (Pmk1-GFP, Pyp1–13myc) and LS139 (pmk1Δ, Pyp1–13myc) (left panel), and LS140 (Pmk1-GFP, Ptc1–13myc) and LS141 (pmk1Δ, Ptc1–13myc) (right panel) were grown to mid log-phase, and either Pyp1 or Ptc1 protein levels were detected by immunoblotting with anti-c-myc antibody. Anti-Cdc2 antibody was used as loading control.

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: Activity Assay, Western Blot, Incubation, Sensitive Assay

Journal: The Journal of Biological Chemistry

Article Title: Biological Significance of Nuclear Localization of Mitogen-activated Protein Kinase Pmk1 in Fission Yeast

doi: 10.1074/jbc.M112.345611

Figure Lengend Snippet: Stress-induced phosphorylation of membrane-targeted Pmk1. A, strains LS111 (Pmk1-GFP; control) and LS115 (Pmk1-GFP-CAAX) were grown in YES medium to mid log-phase and treated with 1 mm hydrogen peroxide. At timed intervals either activated or total Pmk1 were detected by immunoblotting with anti-phospho-p42/44 or anti-GFP antibodies, respectively. B, both activated and total Pmk1 were detected in strains LS111 and LS115 after glucose depletion. C, exponentially growing cells from strains LS111 and LS115 (left panel), LS111 and LS137 (Pmk1-GFP-RitC; middle panel), or LS132 (sty1Δ, Pmk1-GFP) and LS133 (sty1Δ, Pmk1-GFP-CAAX; right panel) were treated with 0.6 m potassium chloride, and both activated and total Pmk1 were detected as described above.

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: Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Biological Significance of Nuclear Localization of Mitogen-activated Protein Kinase Pmk1 in Fission Yeast

doi: 10.1074/jbc.M112.345611

Figure Lengend Snippet: Membrane-tethered Pmk1 promotes vacuole fusion induced during hypotonic stress. A, strains LS111 (Pmk1-GFP; control) and LS115 (Pmk1-GFP-CAAX) were grown in YES medium plus 0.8 m sorbitol to mid log-phase and shifted to the same medium without polyol. At timed intervals either activated or total Pmk1 was detected by immunoblotting with anti-phospho-p42/44 or anti-GFP antibodies, respectively. B, vacuole fusion is shown. Strains LS111 (Pmk1-GFP; control), MI102 (pmk1Δ), LS115 (Pmk1-GFP-CAAX), and MI212 (pmp1Δ) were grown in YES medium plus 0.8 m sorbitol. Cells were labeled with CDCFDA and resuspended in YES medium, and vacuole size and fusion visualized by fluorescence microscopy. The average number of vacuoles per cell is shown as an insert for control and mutant strains.

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: Western Blot, Labeling, Fluorescence, Microscopy, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Biological Significance of Nuclear Localization of Mitogen-activated Protein Kinase Pmk1 in Fission Yeast

doi: 10.1074/jbc.M112.345611

Figure Lengend Snippet: Cell separation defect in cells expressing a Pmk1 version excluded from the nucleus. A, strains LS111 (Pmk1-GFP; Control), MI102 (pmk1Δ), LS115 (Pmk1-GFP-CAAX), and LS137 (Pmk1-GFP-RitC) were grown in YES medium plus 0.8 m sorbitol to mid log-phase, and the percentage of unseptated/septated cells in cultures was determined by fluorescence microscopy after cell staining (n≥400) with calcofluor white. B, representative cells from the strains described above as observed by fluorescence microscopy. C, cells from strains LS123 (cdc25-22, Pmk1-GFP), LS124 (cdc25-22, Pmk1-GFP-CAAX), and MI601 (cdc25-22, pmk1Δ) were grown to A600 = 0.3 at 25 °C, shifted to 37 °C for 3.5 h, and then released from the growth arrest by transfer back to 25 °C. Aliquots were taken at different time intervals, and activated Pmk1 was detected by immunoblotting with anti-phospho-p42/44 antibodies. Anti-Cdc2 antibody was employed as loading control. Percentage of unseptated, septated, and multiseptated cells at each time point is shown as described in A.

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, Fluorescence, Microscopy, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Biological Significance of Nuclear Localization of Mitogen-activated Protein Kinase Pmk1 in Fission Yeast

doi: 10.1074/jbc.M112.345611

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

Journal: The Journal of Biological Chemistry

Article Title: Biological Significance of Nuclear Localization of Mitogen-activated Protein Kinase Pmk1 in Fission Yeast

doi: 10.1074/jbc.M112.345611

Figure Lengend Snippet: Biological relevance of increased nuclear localization of Pmk1. A, shown is a scheme representing a version of Pmk1 fused at its C terminus to the SV40 nuclear localization signal followed by GFP epitope and growing cells of strain LS131 expressing the Pmk1-NLS-GFP fusion as seen by fluorescence microscopy. B, exponentially growing cells of strains LS111 (Pmk1-GFP, control) and LS131 (Pmk1-NLS-GFP) were treated with 0.6 m potassium chloride, and both activated and total Pmk1 were detected by immunoblotting with anti-phospho-p42/44 or anti-GFP antibodies, respectively. C, vic phenotype and chloride sensitivity assays for strains LS111 (control), MI102 (pmk1Δ), LS115, and LS131 are shown. After growth in YES medium, 104, 103, 102, or 10 cells were spotted onto YES plates supplemented with 0.2 m MgCl2 plus 0.5 μg/ml FK506 (vic), or 0.25 m MgCl2 alone and incubated for 3 days at 28 °C before being photographed. D, shown are caspofungin sensitivity assays for the strains described in C.

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, Fluorescence, Microscopy, Western Blot, Incubation