pgk1 mouse mab  (Santa Cruz Biotechnology)

Journal: eLife

Article Title: Glycolysis-dependent sulfur metabolism orchestrates morphological plasticity and virulence in fungi

doi: 10.7554/eLife.109075

Figure Lengend Snippet: ( A ) Epitope-tagged strains of various proteins involved in the de novo biosynthesis of sulfur-containing amino acids, including Met4, Met32, Met16, Met10, Cys4, and Cys3 were spotted on SLAD in the presence and absence of sub-inhibitory concentration of 2-Deoxy-D-Glucose (2DG) following which, cells from colonies were isolated at day 5 and levels of these proteins were assessed using Western blotting. Raw images of Western blots were analyzed using ImageJ to normalize targeted protein expression with the expression of housekeeping protein- β-actin, in order to generate densitometric graphs. Statistical analysis was done using unpaired t-test, **(p<0.01) and *(p<0.05). Error bars represent SEM. ( B ) Epitope-tagged strains of various proteins involved in the de novo biosynthesis of sulfur-containing amino acids, including Met32, Met16, Met10, and Cys3 were grown in liquid SLAD and then treated with sub-inhibitory concentration of 2DG for 24 hr after which protein expression was checked using Western blotting. Pgk1 was used as loading control. ( C ) Raw images of Western blots were analyzed using ImageJ to normalize targeted protein expression with the expression of housekeeping protein-Pgk1, in order to generate densitometric graphs. Statistical analysis was done using unpaired t-test, ***(p<0.001), **(p<0.01) and *(p<0.05). Error bars represent SEM. This figure was created using Biorender.com. Figure 2—figure supplement 2—source data 1. Uncropped and labeled blots for . Figure 2—figure supplement 2—source data 2. Raw unedited blots for .

Article Snippet: Pgk1 Mouse mAb (Cat. #sc-130335) was purchased from Santa Cruz Biotechnology.

Techniques: Concentration Assay, Isolation, Western Blot, Expressing, Control, Labeling

Journal: eLife

Article Title: Metabolic regulation of misfolded protein import into mitochondria

doi: 10.7554/eLife.87518

Figure Lengend Snippet: ( A ) Representative super-resolution imaging for FlucSM split-GFP (spGFP) signal in mitochondria after 90 min estradiol induction. Top, maximum projection. Bottom, 3D rendering. OM: outer membrane labeled by Tom70-mCherry. ( B, C ) Representative images ( B ) and quantification ( C ) of time-dependent accumulation of FlucSM spGFP signal in mitochondria at 30°C after estradiol or ethanol treatment in HG medium. Shown in ( B ): top, FlucSM spGFP; middle: mitochondria labeled with Tom70-mCherry; bottom: merged images. Shown in ( C ): means ± SEM of spGFP intensity (n=3). Paired two-tailed t -test comparing 0 min and 90 min estradiol treatment. ( D, E ) Representative images ( D ) and quantification ( E ) of FlucWT, FlucSM, and FlucDM spGFP after 90 min estradiol treatment in HG medium. Shown in ( E ): means ± SEM of normalized spGFP intensities (n=4). Unpaired two-tailed t -test between FlucWT and FlucDM. ( F, G ) Representative images ( F ) and quantification ( G ) of FlucSM spGFP in cells that grew in HG, LG, and LG-Gly media. Shown in ( G ): means ± SEM of spGFP intensity (n=3). Paired two-tailed t -test. ( H, I ) Representative images ( H ) and quantification ( I ) of the nuclear-cytoplasmic translocation of Mig1-GFP. Shown in ( I ): means ± SEM of normalized Mig-GFP nuclear-cytoplasmic ratio (n=3). Paired (wild-type [WT] in HG vs. LG or LG-Gly) or unpaired (WT vs. Δreg1 in HG) two-tailed t -test, and one-way ANOVA for comparing Δreg1 in HG, WT in LG, and WT in LG-Gly. ( J ) Immunoblots of FlucSM-HA-GFP 11 and Grx5-HA-GFP 1-10 in cell lysates. Relative FlucSM levels (FlucSM/Pgk1) and relative Grx5 levels (matured Grx5/Pgk1) of three biological repeats are shown. Paired (HG vs. LG or LG-Gly) or unpaired (WT vs. Δreg1 in HG) two-tailed t -test. Asterisks indicate the precursor species of Grx5-HA-GFP 1-10 that are excluded from quantification. HG: 2% glucose; LG: 0.1% glucose plus 3% glycerol; LG-Gly: 0.1% glucose. EtOH: ethanol. *p<0.05; **p<0.01; ***p<0.001; ns, not significant, p>0.05. Scale bars, 5 μm. Figure 1—figure supplement 2—source data 1. Raw data for split-GFP (spGFP) intensity and Mig1-GFP quantification. Figure 1—figure supplement 2—source data 2. Raw and labeled immunoblots for .

Article Snippet: PGK1 mouse mAb (22C5D8) was purchased from Invitrogen/Thermo Fisher Scientific.

Techniques: Imaging, Membrane, Labeling, Two Tailed Test, Translocation Assay, Western Blot

Journal: Cell Reports Methods

Article Title: Protein fluorescent labeling in live yeast cells using scFv-based probes

doi: 10.1016/j.crmeth.2022.100357

Figure Lengend Snippet: Labeling of Rfa1-HA by 2E2-NLS-Envy maintains Rfa1 functionality and leads to enhanced detection of RPA foci (A) Phenotypic analysis of yeast strains expressing different versions of tagged Rfa1 including strains expressing Rfa1-3HA or Rfa1-6HA and 2E2-NLS-Envy expressed from the weak URA3 promoter. Spot growth assay on YPD agar plates with or without MMS indicates a phenotypic defect in a strain expressing C′-terminal Rfa1-Envy grown in the presence of MMS. (B) Representative images of RPA foci in cells expressing Rfa1-Envy or Rfa1-HA labeled with 2E2-NLS-Envy, following DNA-damage induction. Lower nuclear fluorescent background is observed in labeled Rfa1-HA cells relative to Rfa1-Envy cells (see Figure S2 E for quantification). Z-plane orthogonal projections of 3D cell images are shown. (C) Enhanced detection of RPA foci upon DNA-damage induction in strains expressing Rfa1-HA labeled with 2E2-NLS-Envy relative to the Rfa1-Envy strain. Rfa1-HA labeling with 2E2-Envy without NLS did not lead to enhanced detection of RPA foci relative to Rfa1-Envy. Results represent an average of three independent repeats, and error bars represent the standard error of the mean (SEM). At least 240 cells were examined in each experiment. (D) Detection of Rfa1 expression in the labeled Rfa1-HA strains using WB analysis showing the dual usage of these strains for microscopy and WB analysis. PGK1 examination is used as an internal control. In all Rfa1-HA-labeled strains, 2E2-Envy is expressed from the weak URA3 promoter.

Article Snippet: Mouse anti-PGK1 mAb , Invitrogen , Cat# 459250; RRID: AB_2532235.

Techniques: Labeling, Expressing, Growth Assay, Microscopy

Journal: Cell Reports Methods

Article Title: Protein fluorescent labeling in live yeast cells using scFv-based probes

doi: 10.1016/j.crmeth.2022.100357

Figure Lengend Snippet:

Article Snippet: Mouse anti-PGK1 mAb , Invitrogen , Cat# 459250; RRID: AB_2532235.

Techniques: Recombinant, Staining, Plasmid Preparation, Microscopy, Western Blot, Amplification, Expressing, Labeling, Software, Stripping Membranes, Transferring, Imaging, Spectrophotometry

Journal: G3: Genes|Genomes|Genetics

Article Title: A case of convergent-gene interference in the budding yeast knockout library causing chromosome instability

doi: 10.1093/g3journal/jkab084

Figure Lengend Snippet: CTF13 mRNA length and 3′ end composition are altered in the sno1Δ mutant. (A) Nested reverse transcriptase (RT)-PCR products from a 3′ RACE assay using a CTF13 gene specific primer (MRG577) and Q I primer (see Supplementary Table S2) run on a 1% agarose gel stained with ethidium bromide. Lanes 1 + 7: 5 μL 1 kb ladder (NEB # N3232L). Lane 2: Control strain, wild-type SNO1-CTF13 locus. Lane 3: sno1Δ::Kan , Lanes 4-6: 3 independent isolates of sno1Δ::Kan-T ADH1 . All strains were engineered into the qCTF background. (B) Sanger sequencing of gel extracted 3′ RACE PCR products for the sno1Δ strain trimmed to a 76 base pair window within the CTF13 3′ UTR. This includes the final base calls from this sequenced fragment. Purple Ns are reads that could not be accurately assigned using the sangerseqR package in R ( Hill et al. 2014 ). Full Sanger sequencing data is available in Figure S5. (C) Graphical representation of the two plasmids used to generate sno1Δ mutants. Plasmid maps are displayed as linear fragments zoomed into the KanMX promoter, gene, and terminator segment. pFA6a-KanMX (top) contains the TEF1 promoter and terminator (P TEF1 and T TEF1 , respectively) sequence from A. gossypii . pFA6a-KanMX-T ADH1 (bottom, referred to as MGP026 in Supplementary Table S4) was cloned using the pFA6a-KanMX as a backbone and the T TEF1 was replaced with a 166 base pair ADH1 terminator (T ADH1 ) sequence from S. cerevisiae . Maps drawn to scale. (D) Sanger sequencing of the 3′ RACE product for the CTF13 mRNA in the sno1Δ::Kan-T ADH1 strain trimmed to a 78 base pair window within the CTF13 3′ UTR. The putative polyA tail signal is shown underlined in black. Full Sanger sequencing results available in Supplementary Figure S6. A black bar is used to indicate the putative polyA reads from this sequencing experiment. (E) qCTF assay CIN rate measurements from three independent isolates of the sno1Δ::Kan-T ADH1 (T ADH1 #1-3) strain and sno1Δ strain. P-values are generated from a Tukey’s post hoc test. (F) qPCR analysis of CTF13 mRNA levels performed on control , sno1Δ , and sno1Δ::Kan-T ADH1 (T ADH1 ) strains using a CTF13 ORF primer sets (set #3, see Supplementary Table S3). Three independent replicates were performed with technical triplicates. Each values is normalized to the level of ACT1 mRNA. Technical triplicates are averaged and pooled together and standard deviation is calculated for means of the independent strains. P-values are generated from a Tukey’s post hoc test. (G) qPCR analysis of CTF13 mRNA levels expressed as fold changes performed on control and sno1Δ::Kan-T ADH1 (labeled T ADH1 for shorthand) in the presence and absence of functional NMD using a CTF13 ORF primer set (set #3, see Supplementary Table S3). Three independent replicates were performed each including technical triplicates. Each values is normalized to the level of ACT1 mRNA. Technical triplicates are averaged and pooled together and standard deviation is calculated for means of the independent strains. Statistical analysis is generated from a Tukey’s post hoc test. (H) Western blot analysis for 3xHA-CTF13 protein levels in a SNO1 WT background or a sno1Δ::Kan-T ADH1 background. Pgk1 was used as a loading control for quantification in Figure 4I . Exposure times were as follows: 5 min and 10 s for α-HA blot, 1 min and 2 s for α-Pgk1 blot. Uncropped membrane images including the molecular weight are provided in Figure S8E). (I) Quantification of the membrane shown in Figure 4H . Ratios between the HA and Pgkl signal were determined using Image Studio Lite. Statistical analysis was performed using a Tukey’s post hoc test and error bars represent the standard deviation.

Article Snippet: Pgk1 mouse monoclonal antibody (1:3000 dilution) and HA rabbit monoclonal antibody (1:1000 dilution) were purchased from Invitrogen (Cat #: 459250) and Cell Signaling (Cat #: 3724S), respectively.

Techniques: Mutagenesis, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Control, Sequencing, Plasmid Preparation, Clone Assay, Generated, Standard Deviation, Labeling, Functional Assay, Western Blot, Membrane, Molecular Weight

Journal:

Article Title: ELA1 and CUL3 Are Required Along with ELC1 for RNA Polymerase II Polyubiquitylation and Degradation in DNA-Damaged Yeast Cells ▿

doi: 10.1128/MCB.00091-07

Figure Lengend Snippet: Rpb1 is not degraded in UV- or 4-NQO-treated ela1Δ or cul3Δ cells. (A) Rpb1 levels in wild-type, ela1Δ, and cul3Δ strains following UV treatment. Yeast cell extracts were prepared at the indicated times after UV irradiation. Rpb1 and the loading control PGK1 were detected by SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with MAb 8WG16 and MAb PGK1, respectively. (B) Rpb1 levels in wild-type, ela1Δ, and cul3Δ strains following treatment with 4-NQO. 4-NQO was added to liquid cultures of log-phase yeast cells at the indicated concentrations, and cells were collected at 60 min after the addition. Cell extracts were prepared, and Rpb1 and PGK1 detected by Western blot analysis as performed for the cell extracts shown in panel A.

Article Snippet: As a loading control, phosphoglycerate kinase 1 (PGK1) levels were examined in the same gel by using mouse MAb PGK1 (Molecular Probes).

Techniques: Irradiation, Polyacrylamide Gel Electrophoresis, Western Blot