Journal: The Journal of Cell Biology

Article Title: Assembly and phosphoregulatory mechanisms of the budding yeast outer kinetochore KMN complex

doi: 10.1083/jcb.202506015

Figure Lengend Snippet: Preparation of S. cerevisiae KMN complex sample for cryo-EM and validation of in vivo protein depletion and in vivo rescue allele expression. (A) Subcomplex and subunit domain organization of the S. cerevisiae KMN complex proteins. CC, CH, HB, N-IDR, and RING-WD40-DEAD box helicase (RWD) domains are highlighted. (B) Coomassie brilliant blue–stained SDS-PAGE gels of the purified KMN subcomplexes. (C–E) Coomassie brilliant blue–stained SDS-PAGE gels of isolated (C) N and (D) K HB-RWD M subcomplexes, or (E) reconstituted K HB-RWD MN complexes after separation using 10–30% glycerol gradients. (F) Coomassie brilliant blue–stained SDS-PAGE gels of the reconstituted K HB-RWD MN complex crosslinked with a 0.0–0.2% glutaraldehyde gradient over the course of a 10–30% glycerol gradient using the GraFix methodology . Crosslinking was repeated twice with identical results. Fractions corresponding to the glycerol gradient volume 2.00–2.75 ml were pooled for cryo-EM sample preparation. Fractions corresponding to the glycerol gradient volume 2.00–2.75 ml were pooled for cryo-EM sample preparation. (G) Immunoblots of WCEs of the indicated yeast strains treated for the indicated duration with PBS or 0.5 mM IAA. Membranes were blotted with anti-FLAG primary antibody to detect NNF1-mAID 3 -FLAG 5 gene products (top panel) or anti-α-tubulin primary antibody as a loading control (bottom panel). (H) Immunoblots of WCEs of the indicated yeast strains treated for the indicated duration with PBS or 0.5 mM IAA. Membranes were blotted with anti-FLAG primary antibody to detect MTW1-mAID 3 -FLAG 5 gene products (top panel) or anti-α-tubulin primary antibody as a loading control (bottom panel). (I) Immunoblots of WCEs of the indicated yeast strains. Membranes were blotted with anti-V5 primary antibodies to detect the gene products expressed from the NNF1 or nnf1 ΔC variant alleles (top panel), or with anti-α-tubulin primary antibody as a loading control (bottom panel). (J) Immunoblots of WCEs of the indicated yeast strains. Membranes were blotted with anti-HA primary antibodies to detect the gene products expressed from the indicated MTW1 or mtw1 ΔC variant alleles (top panel), or with anti-α-tubulin primary antibody as a loading control (bottom panel). CC, coiled coil; HB, helical bundle; WCEs, whole-cell extracts. Source data are available for this figure: .

Article Snippet: To detect alpha tubulin as a loading control, anti-α-tubulin (MCA78G; Bio-Rad, rat monoclonal) antibody was used.

Techniques: Cryo-EM Sample Prep, Biomarker Discovery, In Vivo, Expressing, Staining, SDS Page, Purification, Isolation, Sample Prep, Western Blot, Control, Variant Assay

Journal: PLOS Genetics

Article Title: Inducible gene deletion reveals essentiality of protein kinases and a septation initiation network in Candida albicans

doi: 10.1371/journal.pgen.1012118

Figure Lengend Snippet: Strains with or without Myc-tagged orf19.3456 and HA-tagged orf19.193 were grown to log phase in YPD at 30°C. Protein extracts were prepared as described in materials and methods and immunoprecipitated with an anti-Myc antibody. The immunoprecipitated orf19.3456 -encoded kinase was detected by Western blotting with an anti-Myc antibody, and the co-immunoprecipitated orf19.193 -encoded protein with an anti-HA antibody (top panels, IP). Tagged proteins in the input samples were detected with anti-HA and anti-Myc antibodies (bottom panels, input). Detection of tubulin with an anti-tubulin antibody served as loading control for the input samples. Both independently generated series of strains were used for the experiment.

Article Snippet: For the detection of tubulin, membranes were blocked with 5% milk in TBST and incubated overnight at 4°C with rat anti-tubulin alpha antibody MCA 78G (Bio-Rad), washed with TBST, and then incubated with rabbit anti-rat HRP-conjugated antibody STAR21B (Bio-Rad).

Techniques: Immunoprecipitation, Western Blot, Control, Generated