1nm pp1  (MedChemExpress)

Journal: Life Science Alliance

Article Title: Congress of multiple dimers is needed for cross-phosphorylation of IRE1α and its RNase activity

doi: 10.26508/lsa.202302562

Figure Lengend Snippet: (A) Cells reconstituted with FL-, dim-, dim-DDD- and dim-KB-IRE1 were induced with different concentrations of doxycycline to induce comparable low, medium, and high expression levels, as indicated. IRE1 mutant proteins were revealed by immunoblot with anti-IRE1 antibodies. (B) Cells expressing dim-DDD- and dim-KB-IRE1 at low or high levels were treated with AP20187 (green) and/or 1NM-PP1 (blue) as indicated. Immunoblot analysis with anti-IRE1 antibodies shows that none of the treatments alter the expression levels of either of the two IRE1 mutants. (C) Similar to , HeLa cell lines expressing KB- and KB-DDD-IRE1α mutants under the TetON-inducible promoter were treated with different concentrations of doxycycline to adjust the expression level of the transgenes to comparable levels between each other (low and medium levels in this case). Aliquots from the cell lysates were resolved electrophoretically and the blots decorated with anti-IRE1α. (D) Microscopy images of cells expressing GFP-tagged KB- and KB-DDD-IRE1 at the medium expression level (see (C)). The boxed area in each image is shown magnified on the right, to facilitate the comparison. KB-DDD-IRE1 forms distinct foci upon treatment with Tm and 1NM-PP1, whereas KB-IRE1 does not. All images have been acquired with the same magnification; scale bar (valid for all panels) = 10 μm.

Article Snippet: For allosteric activation of kinase bypass IRE1 mutants (KB-, KB-DDD- and dim-KB-IRE1), 1NM-PP1 (MedChemExpress) was used at the concentration of 7 μM.

Techniques: Expressing, Mutagenesis, Western Blot, Microscopy, Comparison

Journal: Life Science Alliance

Article Title: Congress of multiple dimers is needed for cross-phosphorylation of IRE1α and its RNase activity

doi: 10.26508/lsa.202302562

Figure Lengend Snippet: (A) Cells expressing endogenous IRE1 were treated with dimerizing drug AP20187, nucleotide analog 1NM-PP1, and/or Tm, as indicated. As shown by the XBP1 splicing assay, in none of the conditions AP20187 and/or 1NM-PP1 affected endonuclease activity of wt IRE1. (B) HeLa cells reconstituted with full-length (FL) or dimerizable (dim) IRE1 were treated with doxycycline to achieve low or high expression levels of the respective protein. Cells were then treated with Tm (red) or AP20187 (green). Neither of the treatments alter the expression levels of IRE1, as shown by immunoblot with anti-IRE1 antibodies. A similar result for two other IRE1 mutants is shown in . (C) Cells expressing dim-IRE1 at high or medium levels were treated with increasing concentrations of AP20187 as indicated. As shown by the XBP1 splicing assay, a concentration of 5 nM is already sufficient to activate endonuclease activity if dim-IRE1 is expressed at high levels. On the contrary, if dim-IRE1 is present in lesser amounts (medium expression) AP20187 is unable to trigger XBP1 splicing even at 320 nM concentration. Key for activation is therefore the concentration of IRE1 dimers. In the rest of the study, unless differently stated, we have used AP20187 at a concentration of 10 nM.

Article Snippet: For allosteric activation of kinase bypass IRE1 mutants (KB-, KB-DDD- and dim-KB-IRE1), 1NM-PP1 (MedChemExpress) was used at the concentration of 7 μM.

Techniques: Expressing, Splicing Assay, Activity Assay, Western Blot, Concentration Assay, Activation Assay

Journal: Life Science Alliance

Article Title: Congress of multiple dimers is needed for cross-phosphorylation of IRE1α and its RNase activity

doi: 10.26508/lsa.202302562

Figure Lengend Snippet: (A) Endonuclease activity of dimerizable IRE1α mutants at different expression levels (low, medium, high) after treatment with or without dimerizing drug (AP20187, green) and with or without 1NM-PP1 (blue). (B) Cells expressing FL-, dim-KB-, and dim-DDD-IRE1, at low or high levels as indicated, were treated with Tm (red) or AP20187 (green). The phosphorylation state of IRE1 mutants was assessed by Phos-tag gels and immunoblot with anti-IRE1 antibodies. dim-DDD (because of the substitutions of the three serines) and dim-KB (because of the inability to bind ATP and, hence, phosphotransfer) cannot be phosphorylated even if highly expressed. (C) Endonuclease activity of nucleotide binding requiring (KB-)IRE1α mutants, expressed at low and medium levels, after treatment with Tm (red) and/or NM-PP1 (blue), as indicated. KB-DDD-IRE1α was used to evaluate the possible role of phosphorylation in stabilization of the nucleotide-bound state. XBP1 splicing by both KB mutants depends on nucleotide activation (compare, for instance, lanes 1–2 with 3–4), intracellular concentration (compare lanes 3 with 7 or 4 with 8), and phosphorylation (compare lanes 3 with 11 or 4 with 12). (D) Confocal images of cells expressing GFP-tagged dim-DDD-IRE1. Even at high expression levels the mutant does not form foci upon treatment with AP20187. Scale bar = 10 μm.

Article Snippet: For allosteric activation of kinase bypass IRE1 mutants (KB-, KB-DDD- and dim-KB-IRE1), 1NM-PP1 (MedChemExpress) was used at the concentration of 7 μM.

Techniques: Activity Assay, Expressing, Western Blot, Binding Assay, Activation Assay, Concentration Assay, Mutagenesis

Journal: The EMBO Journal

Article Title: Multi-signal regulation of the GSK-3β homolog Rim11 controls meiosis entry in budding yeast

doi: 10.1038/s44318-024-00149-7

Figure Lengend Snippet: ( A ) Rim11-mNG (FW10297) localization upon rapamycin treatment. Cells were grown in rich medium to exponential growth (exp) and treated with rapamycin for the indicated times. Representative images are shown (left), and nuclear over whole-cell quantification is shown (right). At least n = 50 cells per condition were quantified. Black bars with error bars represent the mean+SD. One-way ANOVA; *** P < 0.001. Scale bar: 5 µm. ( B ) Similar analysis as in A, except that cells were induced to enter meiosis in SPO, SPO + rapamycin, and SPO + glucose. At least n = 50 cells per condition were quantified. Black bars with error bars represent the mean+SD. One-way ANOVA; ** P < 0.01; **** P < 0.0001. Scale bar: 5 µm. ( C ) Similar analysis as in ( A ), except that Rim11-mNG also contained tpk1-as allele described previously (FW10483) was used for the analysis both whole cell and nuclear over whole-cell signals were quantified. Cells were grown in rich medium to exponential growth (exp.) treated with 1NM-PP1, rapamycin or both compounds for 3 h. At least n = 50 cells per condition were quantified. Black bars with error bars represent the mean+SD. One-way ANOVA; *** P < 0.001. Scale bar: 5 µm. ( D – F ) Rim11-mNG localization dynamics during the shift from starvation to rich medium in whole cell, nucleus, and cytoplasm (FW10297). Cells were starved under microfluidics conditions for 24 h before being exposed to synthetic complete medium (SCD) for 4 h. Shown is the Rim11-mNG signal. The data were centered on the starvation to rich medium transition. At least n = 200 cells were quantified. The bold line represents the mean, and the 95% intervals are highlighted by the thin lines. .

Article Snippet: PP1 analog 1NM-PP1 was used to inhibit PKA activity (Calbiochem, Merck Millipore).

Techniques: