anti hog1 antibody  (Santa Cruz Biotechnology)

 
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    Name:
    Hog1 Antibody
    Description:
    Anti Hog1 Antibody D 3 is a mouse monoclonal IgG1 kappa light chain Hog1 antibody provided at 200 µg ml raised against amino acids 291 408 of Hog1 of Saccharomyces cerevisiae origin Hog1 Antibody D 3 is recommended for detection of Hog1 of y origin by WB IP IF and ELISA Anti Hog1 Antibody D 3 is available conjugated to agarose for IP HRP for WB IHC P and ELISA and to either phycoerythrin or FITC for IF IHC P and FCM also available conjugated to Alexa Fluor 488 Alexa Fluor 546 Alexa Fluor 594 or Alexa Fluor 647 for WB RGB IF IHC P and FCM and for use with RGB fluorescent imaging systems such as iBright FL1000 FluorChem Typhoon Azure and other comparable systems also available conjugated to Alexa Fluor 680 or Alexa Fluor 790 for WB NIR IF and FCM for use with Near Infrared NIR detection systems such as LI COR Odyssey iBright FL1000 FluorChem Typhoon Azure and other comparable systems Contact our Technical Service Department or your local Distributor for more information on how to receive a FREE 10 µg sample of Hog1 D 3 sc 165978
    Catalog Number:
    SC-165978
    Price:
    None
    Category:
    Antibodies Primary Antibodies and ImmunoCruz Conjugates Non Mammalian Hog1 Antibodies Hog1 Antibody D 3
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    Structured Review

    Santa Cruz Biotechnology anti hog1 antibody
    Ptc2 inactivates <t>Hog1</t> in vitro. (A) Activated GST-Hog1 was isolated from osmotic stressed yeast and was untreated or treated with Ptc2. Approximately 0.5 μg of GST-Hog1 bound to resin was incubated with 0 to 1.2 μg of Ptc2 for 30 min at 30°C. The resin was washed extensively to remove Ptc2, and GST-Hog1 was incubated with MBP and [γ- 32 P]ATP to assess Hog1 kinase activity. Radiolabel incorporated into MBP was examined by PhosphorImager analysis. (B) Ptc2 inactivates Hog1 by dephosphorylating the phosphothreonine residue (pT) in the activation loop. GST-Hog1 was phosphorylated in vitro, and the sample was untreated (left) or incubated with Ptc2 (right) prior to phosphoamino acid analysis. The radiolabeled amino acids were detected with the PhosphorImager. Arrows, positions of phosphoamino acid standards as revealed by ninhydrin staining.
    Anti Hog1 Antibody D 3 is a mouse monoclonal IgG1 kappa light chain Hog1 antibody provided at 200 µg ml raised against amino acids 291 408 of Hog1 of Saccharomyces cerevisiae origin Hog1 Antibody D 3 is recommended for detection of Hog1 of y origin by WB IP IF and ELISA Anti Hog1 Antibody D 3 is available conjugated to agarose for IP HRP for WB IHC P and ELISA and to either phycoerythrin or FITC for IF IHC P and FCM also available conjugated to Alexa Fluor 488 Alexa Fluor 546 Alexa Fluor 594 or Alexa Fluor 647 for WB RGB IF IHC P and FCM and for use with RGB fluorescent imaging systems such as iBright FL1000 FluorChem Typhoon Azure and other comparable systems also available conjugated to Alexa Fluor 680 or Alexa Fluor 790 for WB NIR IF and FCM for use with Near Infrared NIR detection systems such as LI COR Odyssey iBright FL1000 FluorChem Typhoon Azure and other comparable systems Contact our Technical Service Department or your local Distributor for more information on how to receive a FREE 10 µg sample of Hog1 D 3 sc 165978
    https://www.bioz.com/result/anti hog1 antibody/product/Santa Cruz Biotechnology
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti hog1 antibody - by Bioz Stars, 2021-09
    96/100 stars

    Images

    1) Product Images from "Role of Ptc2 Type 2C Ser/Thr Phosphatase in Yeast High-Osmolarity Glycerol Pathway Inactivation"

    Article Title: Role of Ptc2 Type 2C Ser/Thr Phosphatase in Yeast High-Osmolarity Glycerol Pathway Inactivation

    Journal: Eukaryotic Cell

    doi: 10.1128/EC.1.6.1032-1040.2002

    Ptc2 inactivates Hog1 in vitro. (A) Activated GST-Hog1 was isolated from osmotic stressed yeast and was untreated or treated with Ptc2. Approximately 0.5 μg of GST-Hog1 bound to resin was incubated with 0 to 1.2 μg of Ptc2 for 30 min at 30°C. The resin was washed extensively to remove Ptc2, and GST-Hog1 was incubated with MBP and [γ- 32 P]ATP to assess Hog1 kinase activity. Radiolabel incorporated into MBP was examined by PhosphorImager analysis. (B) Ptc2 inactivates Hog1 by dephosphorylating the phosphothreonine residue (pT) in the activation loop. GST-Hog1 was phosphorylated in vitro, and the sample was untreated (left) or incubated with Ptc2 (right) prior to phosphoamino acid analysis. The radiolabeled amino acids were detected with the PhosphorImager. Arrows, positions of phosphoamino acid standards as revealed by ninhydrin staining.
    Figure Legend Snippet: Ptc2 inactivates Hog1 in vitro. (A) Activated GST-Hog1 was isolated from osmotic stressed yeast and was untreated or treated with Ptc2. Approximately 0.5 μg of GST-Hog1 bound to resin was incubated with 0 to 1.2 μg of Ptc2 for 30 min at 30°C. The resin was washed extensively to remove Ptc2, and GST-Hog1 was incubated with MBP and [γ- 32 P]ATP to assess Hog1 kinase activity. Radiolabel incorporated into MBP was examined by PhosphorImager analysis. (B) Ptc2 inactivates Hog1 by dephosphorylating the phosphothreonine residue (pT) in the activation loop. GST-Hog1 was phosphorylated in vitro, and the sample was untreated (left) or incubated with Ptc2 (right) prior to phosphoamino acid analysis. The radiolabeled amino acids were detected with the PhosphorImager. Arrows, positions of phosphoamino acid standards as revealed by ninhydrin staining.

    Techniques Used: In Vitro, Isolation, Incubation, Activity Assay, Activation Assay, Phosphoamino Acid Analysis, Staining

    Ptc2 inactivates Hog1 in vivo. (A) Overexpression of PTC2 inhibits osmotic-stress-induced Hog1 activation. Hog1 kinase activity in PTC2 overexpressor IMY105, carrying pKT-PTC2 and pHOG1-ha2, and in the control strain, carrying empty vector pKT and pHOG1-ha2, was examined. Before (time zero) and after exposure to osmotic stress (0.4 M NaCl) for various times, Hog1-HA was immunoprecipitated and incubated with MBP and [γ- 32 P]ATP. Radiolabel incorporated into MBP was examined with the PhosphorImager. The graph shows the means of three independent experiments ± standard errors of the means (SEM). (B) Hog1 is hyperactivated in a strain lacking PTC2 and PTC3 . Hog1 kinase activity was monitored prior to and following osmotic stress in a hog1 Δ strain (IMY100) and in a ptc2 Δ ptc3 Δ hog1 Δ strain (CAY9), each carrying a Hog1-HA-expressing plasmid. Hog1 kinase activity was monitored as described for panel A. The graph shows the means of six independent experiments ± SEM. (C) Ptc2 does not inactivate Pbs2 in vivo. Hog1-pY in the 334 strain carrying a plasmid overexpressing PTC2 or an empty vector was examined. The level of Hog1-pY prior to and following osmotic stress was monitored by immunoblotting with an anti-pY antibody. Total Hog1 protein was examined by blotting with an anti-Hog1 antibody.
    Figure Legend Snippet: Ptc2 inactivates Hog1 in vivo. (A) Overexpression of PTC2 inhibits osmotic-stress-induced Hog1 activation. Hog1 kinase activity in PTC2 overexpressor IMY105, carrying pKT-PTC2 and pHOG1-ha2, and in the control strain, carrying empty vector pKT and pHOG1-ha2, was examined. Before (time zero) and after exposure to osmotic stress (0.4 M NaCl) for various times, Hog1-HA was immunoprecipitated and incubated with MBP and [γ- 32 P]ATP. Radiolabel incorporated into MBP was examined with the PhosphorImager. The graph shows the means of three independent experiments ± standard errors of the means (SEM). (B) Hog1 is hyperactivated in a strain lacking PTC2 and PTC3 . Hog1 kinase activity was monitored prior to and following osmotic stress in a hog1 Δ strain (IMY100) and in a ptc2 Δ ptc3 Δ hog1 Δ strain (CAY9), each carrying a Hog1-HA-expressing plasmid. Hog1 kinase activity was monitored as described for panel A. The graph shows the means of six independent experiments ± SEM. (C) Ptc2 does not inactivate Pbs2 in vivo. Hog1-pY in the 334 strain carrying a plasmid overexpressing PTC2 or an empty vector was examined. The level of Hog1-pY prior to and following osmotic stress was monitored by immunoblotting with an anti-pY antibody. Total Hog1 protein was examined by blotting with an anti-Hog1 antibody.

    Techniques Used: In Vivo, Over Expression, Activation Assay, Activity Assay, Plasmid Preparation, Immunoprecipitation, Incubation, Expressing

    The temperature sensitivity of strains lacking PTC s and PTP2 is due to HOG1. (A) Strains lacking PTC s and PTP2 exhibit growth defects at 37°C. The growth of ptp2 Δ (IMY21a), ptc2 Δ ptc3 Δ (IMY124), ptc3 Δ ptp2 Δ (IMY127b), ptc2 Δ ptc3 Δ ptp2 Δ (IMY128), and wild-type (BBY45) strains on rich medium (yeast extract-peptone-dextrose [YPD]) at 37 and 30°C was compared. (B) The growth defects of the ptc3 Δ ptp2 Δ and ptc2 Δ ptc3 Δ ptp2 Δ strains at 37°C were suppressed by deleting HOG1 . The strains lacking HOG1 were JMY1 ( ptc3 Δ ptp2 Δ hog1 Δ and JMY2 ( ptc2 Δ ptc3 Δ ptp2 Δ hog1 Δ) and were compared on YPD at 37 and 30°C.
    Figure Legend Snippet: The temperature sensitivity of strains lacking PTC s and PTP2 is due to HOG1. (A) Strains lacking PTC s and PTP2 exhibit growth defects at 37°C. The growth of ptp2 Δ (IMY21a), ptc2 Δ ptc3 Δ (IMY124), ptc3 Δ ptp2 Δ (IMY127b), ptc2 Δ ptc3 Δ ptp2 Δ (IMY128), and wild-type (BBY45) strains on rich medium (yeast extract-peptone-dextrose [YPD]) at 37 and 30°C was compared. (B) The growth defects of the ptc3 Δ ptp2 Δ and ptc2 Δ ptc3 Δ ptp2 Δ strains at 37°C were suppressed by deleting HOG1 . The strains lacking HOG1 were JMY1 ( ptc3 Δ ptp2 Δ hog1 Δ and JMY2 ( ptc2 Δ ptc3 Δ ptp2 Δ hog1 Δ) and were compared on YPD at 37 and 30°C.

    Techniques Used:

    2) Product Images from "The Sensor Proteins BcSho1 and BcSln1 Are Involved in, Though Not Essential to, Vegetative Differentiation, Pathogenicity and Osmotic Stress Tolerance in Botrytis cinerea"

    Article Title: The Sensor Proteins BcSho1 and BcSln1 Are Involved in, Though Not Essential to, Vegetative Differentiation, Pathogenicity and Osmotic Stress Tolerance in Botrytis cinerea

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.00328

    Model of the yeast hyperosmotic-response MAPK pathway. (A) The osmotic signals from Sho1 or Sln1 are transduced by unique components and converge to activate Pbs2. The Sho1 branch requires Cdc42, Ste20, and Ste50 to activate Ste11. The Sln1 protein activates Ssk2 and Ssk22 through Ypd1 and Ssk1. Any of Ste11, Ssk2, or Ssk22 is able to activate Pbs2, which then phosphorylates Hog1, resulting in translocation of Hog1 to the nucleus that regulates responsible genes expression for osmoadaptation. (B) The schematic diagram of Sho1 and Sln1 orthologs in Botrytis cinerea . The functional domains were retrieved in EnsemblFungi ( https://fungi.ensembl.org/Botrytis_cinerea/Info/Index ). (C) Subcellular localization of BcSho1-GFP and BcSln1-GFP fusion proteins in B. cienrea . Bars, 10 μm.
    Figure Legend Snippet: Model of the yeast hyperosmotic-response MAPK pathway. (A) The osmotic signals from Sho1 or Sln1 are transduced by unique components and converge to activate Pbs2. The Sho1 branch requires Cdc42, Ste20, and Ste50 to activate Ste11. The Sln1 protein activates Ssk2 and Ssk22 through Ypd1 and Ssk1. Any of Ste11, Ssk2, or Ssk22 is able to activate Pbs2, which then phosphorylates Hog1, resulting in translocation of Hog1 to the nucleus that regulates responsible genes expression for osmoadaptation. (B) The schematic diagram of Sho1 and Sln1 orthologs in Botrytis cinerea . The functional domains were retrieved in EnsemblFungi ( https://fungi.ensembl.org/Botrytis_cinerea/Info/Index ). (C) Subcellular localization of BcSho1-GFP and BcSln1-GFP fusion proteins in B. cienrea . Bars, 10 μm.

    Techniques Used: Translocation Assay, Expressing, Functional Assay

    3) Product Images from "A Single MAPKKK Regulates the Hog1 MAPK Pathway in the Pathogenic Fungus Candida albicans"

    Article Title: A Single MAPKKK Regulates the Hog1 MAPK Pathway in the Pathogenic Fungus Candida albicans

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E07-06-0581

    ssk2Δ and hog1 Δ cells display similar phenotypes. (A) Approximately 10 3 cells, and 10-fold dilutions thereof, of exponentially growing wild-type ( Wt -BWP17), hog1 Δ (JC47), ssk2 Δ (JC482), ssk2 Δ+ SSK2 (JC620), ste11
    Figure Legend Snippet: ssk2Δ and hog1 Δ cells display similar phenotypes. (A) Approximately 10 3 cells, and 10-fold dilutions thereof, of exponentially growing wild-type ( Wt -BWP17), hog1 Δ (JC47), ssk2 Δ (JC482), ssk2 Δ+ SSK2 (JC620), ste11

    Techniques Used:

    CaSsk2 is not essential for the interaction of Hog1 with Pbs2. Coprecipitation experiments to examine the association of Hog1 with Pbs2 in wild-type and ssk2 Δ cells. Extracts were prepared from mid-log cultures of Wt (BWP17) and ssk2 Δ
    Figure Legend Snippet: CaSsk2 is not essential for the interaction of Hog1 with Pbs2. Coprecipitation experiments to examine the association of Hog1 with Pbs2 in wild-type and ssk2 Δ cells. Extracts were prepared from mid-log cultures of Wt (BWP17) and ssk2 Δ

    Techniques Used:

    CaSsk2 is required for the activation of Hog1-dependent gene expression. (A) Northern blot analysis of RNA isolated from mid-log cultures of Wt (BWP17), hog1 Δ (JC47), and ssk2 Δ (JC482) cells treated with either 5 mM H 2 O 2 or 0.3 M NaCl
    Figure Legend Snippet: CaSsk2 is required for the activation of Hog1-dependent gene expression. (A) Northern blot analysis of RNA isolated from mid-log cultures of Wt (BWP17), hog1 Δ (JC47), and ssk2 Δ (JC482) cells treated with either 5 mM H 2 O 2 or 0.3 M NaCl

    Techniques Used: Activation Assay, Expressing, Northern Blot, Isolation

    CaSsk2 but not CaSte11 is required for Hog1 phosphorylation. (A) Western blot analysis of whole cell extracts isolated from wild-type ( Wt -BWP17), ssk2 Δ (JC482), ssk2 Δ+ SSK2 (JC620), and ste11 Δ (JC524) cells after treatment with
    Figure Legend Snippet: CaSsk2 but not CaSte11 is required for Hog1 phosphorylation. (A) Western blot analysis of whole cell extracts isolated from wild-type ( Wt -BWP17), ssk2 Δ (JC482), ssk2 Δ+ SSK2 (JC620), and ste11 Δ (JC524) cells after treatment with

    Techniques Used: Western Blot, Isolation

    CaSsk2 but not CaSte11 is required for Hog1 nuclear localization. The localization of YFP-tagged Hog1 was determined by fluorescence microscopy in wild-type ( Wt -JC54), ssk2 Δ (JC522), and ste11 Δ (JC545) cells before stress and after treatment
    Figure Legend Snippet: CaSsk2 but not CaSte11 is required for Hog1 nuclear localization. The localization of YFP-tagged Hog1 was determined by fluorescence microscopy in wild-type ( Wt -JC54), ssk2 Δ (JC522), and ste11 Δ (JC545) cells before stress and after treatment

    Techniques Used: Fluorescence, Microscopy

    Model depicting the relay of osmotic and oxidative stress signals to the Hog1 MAPK in C. albicans
    Figure Legend Snippet: Model depicting the relay of osmotic and oxidative stress signals to the Hog1 MAPK in C. albicans

    Techniques Used:

    4) Product Images from "Mitochondrial versus nuclear gene expression and membrane protein assembly: the case of subunit 2 of yeast cytochrome c oxidase"

    Article Title: Mitochondrial versus nuclear gene expression and membrane protein assembly: the case of subunit 2 of yeast cytochrome c oxidase

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-09-0560

    C c O complex activity remains unchanged when the mitochondria-encoded Cox2 subunit contains the W56R mutation. (A) Yeast cytosolic (c) and mitochondrial (m) fractions (100 and 50 µg of protein, respectively) of the indicated strains were separated by SDS-Tricine-PAGE and transferred to nitrocellulose membranes for Western blot analysis with an anti-Cox2 antibody (top panel). Bands corresponding to the mature Cox2 subunits and the cCox2 W56R precursor (still retaining the MTS of Oxa1) are indicated. Parallel Western blots with antibodies against cytosolic Hog1 and against the mitochondrial proteins Atp2 and Oxa1 served as loading and cell fractionation controls (bottom panels). (B) Mitochondrial fractions from A were treated with 100 µg/ml Proteinase K (PK). The asterisk indicates a partial degradation of Cox2, possibly due to imperfect preparation of the mitochondrial fractions. Bands corresponding to the mature Cox2 subunits are indicated (m), as well as that of the remaining precursor (p). (C) Tenfold serial dilutions from yeast cultures were spotted on fermentative (glucose) or on nonfermentative (ethanol/glycerol) plates in the presence and absence of 5 µM Cu, Co, and Mg bivalent salts at 30 and 37°C. A Δcoa6 strain was included as a positive control for growth rescue by copper supplementation. (D) Isolated mitochondria (250 µg) from the indicated strains were solubilized with lauryl maltoside and separated by BN–PAGE (4–15%). Lanes 1–4, C c O in gel activity; lanes 5–8, ATPase activity used as loading control. Bands corresponding to C c O (IV) and the F 1 Fo-ATPase (V) are indicated. Band IV* corresponds to C c O lacking the Cox6 subunit ( Horan et al. , 2005 ). (E) Mitochondria from D were solubilized with digitonin, separated by BN–PAGE (4–15%), and stained for C c O in gel activity. Bands corresponding to C c O and its supercomplexes are indicated. (F) Oxygen consumption of fasted yeast cells. Arrows indicate where 3 × 10 7 cells were added to the oxygen-meter chamber, 50 mM ethanol was added as substrate, and oxygen uptake was inhibited with 200 µM sodium cyanide. The final volume was 1 ml. The mean and the SD of triplicates are shown for each point.
    Figure Legend Snippet: C c O complex activity remains unchanged when the mitochondria-encoded Cox2 subunit contains the W56R mutation. (A) Yeast cytosolic (c) and mitochondrial (m) fractions (100 and 50 µg of protein, respectively) of the indicated strains were separated by SDS-Tricine-PAGE and transferred to nitrocellulose membranes for Western blot analysis with an anti-Cox2 antibody (top panel). Bands corresponding to the mature Cox2 subunits and the cCox2 W56R precursor (still retaining the MTS of Oxa1) are indicated. Parallel Western blots with antibodies against cytosolic Hog1 and against the mitochondrial proteins Atp2 and Oxa1 served as loading and cell fractionation controls (bottom panels). (B) Mitochondrial fractions from A were treated with 100 µg/ml Proteinase K (PK). The asterisk indicates a partial degradation of Cox2, possibly due to imperfect preparation of the mitochondrial fractions. Bands corresponding to the mature Cox2 subunits are indicated (m), as well as that of the remaining precursor (p). (C) Tenfold serial dilutions from yeast cultures were spotted on fermentative (glucose) or on nonfermentative (ethanol/glycerol) plates in the presence and absence of 5 µM Cu, Co, and Mg bivalent salts at 30 and 37°C. A Δcoa6 strain was included as a positive control for growth rescue by copper supplementation. (D) Isolated mitochondria (250 µg) from the indicated strains were solubilized with lauryl maltoside and separated by BN–PAGE (4–15%). Lanes 1–4, C c O in gel activity; lanes 5–8, ATPase activity used as loading control. Bands corresponding to C c O (IV) and the F 1 Fo-ATPase (V) are indicated. Band IV* corresponds to C c O lacking the Cox6 subunit ( Horan et al. , 2005 ). (E) Mitochondria from D were solubilized with digitonin, separated by BN–PAGE (4–15%), and stained for C c O in gel activity. Bands corresponding to C c O and its supercomplexes are indicated. (F) Oxygen consumption of fasted yeast cells. Arrows indicate where 3 × 10 7 cells were added to the oxygen-meter chamber, 50 mM ethanol was added as substrate, and oxygen uptake was inhibited with 200 µM sodium cyanide. The final volume was 1 ml. The mean and the SD of triplicates are shown for each point.

    Techniques Used: Activity Assay, Mutagenesis, Polyacrylamide Gel Electrophoresis, Western Blot, Cell Fractionation, Positive Control, Isolation, Staining

    5) Product Images from "A Single MAPKKK Regulates the Hog1 MAPK Pathway in the Pathogenic Fungus Candida albicans"

    Article Title: A Single MAPKKK Regulates the Hog1 MAPK Pathway in the Pathogenic Fungus Candida albicans

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E07-06-0581

    ssk2Δ and hog1 Δ cells display similar phenotypes. (A) Approximately 10 3 cells, and 10-fold dilutions thereof, of exponentially growing wild-type ( Wt -BWP17), hog1 Δ (JC47), ssk2 Δ (JC482), ssk2 Δ+ SSK2 (JC620), ste11
    Figure Legend Snippet: ssk2Δ and hog1 Δ cells display similar phenotypes. (A) Approximately 10 3 cells, and 10-fold dilutions thereof, of exponentially growing wild-type ( Wt -BWP17), hog1 Δ (JC47), ssk2 Δ (JC482), ssk2 Δ+ SSK2 (JC620), ste11

    Techniques Used:

    CaSsk2 is not essential for the interaction of Hog1 with Pbs2. Coprecipitation experiments to examine the association of Hog1 with Pbs2 in wild-type and ssk2 Δ cells. Extracts were prepared from mid-log cultures of Wt (BWP17) and ssk2 Δ
    Figure Legend Snippet: CaSsk2 is not essential for the interaction of Hog1 with Pbs2. Coprecipitation experiments to examine the association of Hog1 with Pbs2 in wild-type and ssk2 Δ cells. Extracts were prepared from mid-log cultures of Wt (BWP17) and ssk2 Δ

    Techniques Used:

    CaSsk2 is required for the activation of Hog1-dependent gene expression. (A) Northern blot analysis of RNA isolated from mid-log cultures of Wt (BWP17), hog1 Δ (JC47), and ssk2 Δ (JC482) cells treated with either 5 mM H 2 O 2 or 0.3 M NaCl
    Figure Legend Snippet: CaSsk2 is required for the activation of Hog1-dependent gene expression. (A) Northern blot analysis of RNA isolated from mid-log cultures of Wt (BWP17), hog1 Δ (JC47), and ssk2 Δ (JC482) cells treated with either 5 mM H 2 O 2 or 0.3 M NaCl

    Techniques Used: Activation Assay, Expressing, Northern Blot, Isolation

    CaSsk2 but not CaSte11 is required for Hog1 phosphorylation. (A) Western blot analysis of whole cell extracts isolated from wild-type ( Wt -BWP17), ssk2 Δ (JC482), ssk2 Δ+ SSK2 (JC620), and ste11 Δ (JC524) cells after treatment with
    Figure Legend Snippet: CaSsk2 but not CaSte11 is required for Hog1 phosphorylation. (A) Western blot analysis of whole cell extracts isolated from wild-type ( Wt -BWP17), ssk2 Δ (JC482), ssk2 Δ+ SSK2 (JC620), and ste11 Δ (JC524) cells after treatment with

    Techniques Used: Western Blot, Isolation

    CaSsk2 but not CaSte11 is required for Hog1 nuclear localization. The localization of YFP-tagged Hog1 was determined by fluorescence microscopy in wild-type ( Wt -JC54), ssk2 Δ (JC522), and ste11 Δ (JC545) cells before stress and after treatment
    Figure Legend Snippet: CaSsk2 but not CaSte11 is required for Hog1 nuclear localization. The localization of YFP-tagged Hog1 was determined by fluorescence microscopy in wild-type ( Wt -JC54), ssk2 Δ (JC522), and ste11 Δ (JC545) cells before stress and after treatment

    Techniques Used: Fluorescence, Microscopy

    Model depicting the relay of osmotic and oxidative stress signals to the Hog1 MAPK in C. albicans
    Figure Legend Snippet: Model depicting the relay of osmotic and oxidative stress signals to the Hog1 MAPK in C. albicans

    Techniques Used:

    6) Product Images from "Acetylation of BcHpt Lysine 161 Regulates Botrytis cinerea Sensitivity to Fungicides, Multistress Adaptation and Virulence"

    Article Title: Acetylation of BcHpt Lysine 161 Regulates Botrytis cinerea Sensitivity to Fungicides, Multistress Adaptation and Virulence

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.02965

    BcHpt Lys161 affects the phosphorylation levels of BcSak1. Comparison of BcSak1 phosphorylation in B05.10, ΔBcHpt + BcHpt K161Q -GFP and ΔBcHpt + BcHpt K161R -GFP. Phosphorylated and total BcSak1 proteins were detected using anti-phosphorylated p38 (Thr180/Tyr182) and anti-Hog1 antibodies, respectively.
    Figure Legend Snippet: BcHpt Lys161 affects the phosphorylation levels of BcSak1. Comparison of BcSak1 phosphorylation in B05.10, ΔBcHpt + BcHpt K161Q -GFP and ΔBcHpt + BcHpt K161R -GFP. Phosphorylated and total BcSak1 proteins were detected using anti-phosphorylated p38 (Thr180/Tyr182) and anti-Hog1 antibodies, respectively.

    Techniques Used:

    7) Product Images from "A Single MAPKKK Regulates the Hog1 MAPK Pathway in the Pathogenic Fungus Candida albicans"

    Article Title: A Single MAPKKK Regulates the Hog1 MAPK Pathway in the Pathogenic Fungus Candida albicans

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E07-06-0581

    ssk2Δ and hog1 Δ cells display similar phenotypes. (A) Approximately 10 3 cells, and 10-fold dilutions thereof, of exponentially growing wild-type ( Wt -BWP17), hog1 Δ (JC47), ssk2 Δ (JC482), ssk2 Δ+ SSK2 (JC620), ste11
    Figure Legend Snippet: ssk2Δ and hog1 Δ cells display similar phenotypes. (A) Approximately 10 3 cells, and 10-fold dilutions thereof, of exponentially growing wild-type ( Wt -BWP17), hog1 Δ (JC47), ssk2 Δ (JC482), ssk2 Δ+ SSK2 (JC620), ste11

    Techniques Used:

    CaSsk2 is not essential for the interaction of Hog1 with Pbs2. Coprecipitation experiments to examine the association of Hog1 with Pbs2 in wild-type and ssk2 Δ cells. Extracts were prepared from mid-log cultures of Wt (BWP17) and ssk2 Δ
    Figure Legend Snippet: CaSsk2 is not essential for the interaction of Hog1 with Pbs2. Coprecipitation experiments to examine the association of Hog1 with Pbs2 in wild-type and ssk2 Δ cells. Extracts were prepared from mid-log cultures of Wt (BWP17) and ssk2 Δ

    Techniques Used:

    CaSsk2 is required for the activation of Hog1-dependent gene expression. (A) Northern blot analysis of RNA isolated from mid-log cultures of Wt (BWP17), hog1 Δ (JC47), and ssk2 Δ (JC482) cells treated with either 5 mM H 2 O 2 or 0.3 M NaCl
    Figure Legend Snippet: CaSsk2 is required for the activation of Hog1-dependent gene expression. (A) Northern blot analysis of RNA isolated from mid-log cultures of Wt (BWP17), hog1 Δ (JC47), and ssk2 Δ (JC482) cells treated with either 5 mM H 2 O 2 or 0.3 M NaCl

    Techniques Used: Activation Assay, Expressing, Northern Blot, Isolation

    CaSsk2 but not CaSte11 is required for Hog1 phosphorylation. (A) Western blot analysis of whole cell extracts isolated from wild-type ( Wt -BWP17), ssk2 Δ (JC482), ssk2 Δ+ SSK2 (JC620), and ste11 Δ (JC524) cells after treatment with
    Figure Legend Snippet: CaSsk2 but not CaSte11 is required for Hog1 phosphorylation. (A) Western blot analysis of whole cell extracts isolated from wild-type ( Wt -BWP17), ssk2 Δ (JC482), ssk2 Δ+ SSK2 (JC620), and ste11 Δ (JC524) cells after treatment with

    Techniques Used: Western Blot, Isolation

    CaSsk2 but not CaSte11 is required for Hog1 nuclear localization. The localization of YFP-tagged Hog1 was determined by fluorescence microscopy in wild-type ( Wt -JC54), ssk2 Δ (JC522), and ste11 Δ (JC545) cells before stress and after treatment
    Figure Legend Snippet: CaSsk2 but not CaSte11 is required for Hog1 nuclear localization. The localization of YFP-tagged Hog1 was determined by fluorescence microscopy in wild-type ( Wt -JC54), ssk2 Δ (JC522), and ste11 Δ (JC545) cells before stress and after treatment

    Techniques Used: Fluorescence, Microscopy

    Model depicting the relay of osmotic and oxidative stress signals to the Hog1 MAPK in C. albicans
    Figure Legend Snippet: Model depicting the relay of osmotic and oxidative stress signals to the Hog1 MAPK in C. albicans

    Techniques Used:

    8) Product Images from "Hsp90 Orchestrates Transcriptional Regulation by Hsf1 and Cell Wall Remodelling by MAPK Signalling during Thermal Adaptation in a Pathogenic Yeast"

    Article Title: Hsp90 Orchestrates Transcriptional Regulation by Hsf1 and Cell Wall Remodelling by MAPK Signalling during Thermal Adaptation in a Pathogenic Yeast

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003069

    The cross-talk between thermal adaptation and cell wall stress resistance is not mediated via stress cross-protection. (A) Stress cross-protection in C. albicans wild-type cells (NGY152: Table 1 ) was not observed for cells pre-treated with a heat stress and then subjected to cell wall stress, but was observed for cells exposed to a secondary oxidative stress. The data represent cell survival after exposure to a 30°C–42°C heat shock followed by a subsequent cell wall (CFW, CR), osmotic (NaCl) or peroxide (H 2 O 2 ) stress (see Materials and Methods ) and the data are expressed relative to unstressed cells (dark bars). Control cells (grey bars), were not exposed to the prior 30°C–42°C heat shock. (B) The reciprocal assay was performed, whereby C. albicans wild type cells were exposed to a prior stress (cell wall: CFW, CR; osmotic: NaCl; peroxide: H 2 O 2 ) followed by a 30°C–42°C heat shock (dark bars). These data are expressed relative to unstressed cells. Control cells (grey bars) correspond to cells exposed only to the 30°C–42°C heat shock. (C) Wild type, hog1 Δ (JC50) or cap1 Δ cells (JC128: Table 1 ) were pre-treated with a 30°C–42°C heat shock, followed by a H 2 O 2 stress (dark bars). Control cells (grey bars) were not exposed to the 30°C–42°C heat shock. The data represent the level of survival compared to unstressed cells. All data are the means from three independent assays: ** paired, two-tailed t-test, p
    Figure Legend Snippet: The cross-talk between thermal adaptation and cell wall stress resistance is not mediated via stress cross-protection. (A) Stress cross-protection in C. albicans wild-type cells (NGY152: Table 1 ) was not observed for cells pre-treated with a heat stress and then subjected to cell wall stress, but was observed for cells exposed to a secondary oxidative stress. The data represent cell survival after exposure to a 30°C–42°C heat shock followed by a subsequent cell wall (CFW, CR), osmotic (NaCl) or peroxide (H 2 O 2 ) stress (see Materials and Methods ) and the data are expressed relative to unstressed cells (dark bars). Control cells (grey bars), were not exposed to the prior 30°C–42°C heat shock. (B) The reciprocal assay was performed, whereby C. albicans wild type cells were exposed to a prior stress (cell wall: CFW, CR; osmotic: NaCl; peroxide: H 2 O 2 ) followed by a 30°C–42°C heat shock (dark bars). These data are expressed relative to unstressed cells. Control cells (grey bars) correspond to cells exposed only to the 30°C–42°C heat shock. (C) Wild type, hog1 Δ (JC50) or cap1 Δ cells (JC128: Table 1 ) were pre-treated with a 30°C–42°C heat shock, followed by a H 2 O 2 stress (dark bars). Control cells (grey bars) were not exposed to the 30°C–42°C heat shock. The data represent the level of survival compared to unstressed cells. All data are the means from three independent assays: ** paired, two-tailed t-test, p

    Techniques Used: Two Tailed Test

    Hsp90 coordinates the activities of multiple signalling pathways that contribute to thermotolerance in C. albicans - a model. Hsf1 activation is required for thermotolerance [42] . Hog1, Mkc1 and Cek1 signalling are also required for thermotolerance ( Figure 3 ), but these MAP kinases are not essential for Hsf1 phosphorylation ( Figure 4 ). Instead, these pathways promote thermotolerance in part via cell wall remodelling [81] , [90] . Hsp90 coordinates much of this activity. Hsf1 ( Figures 1 and 2 ), Hog1, Mkc1 and Cek1 ( Figure 10 ) are all Hsp90 client proteins [62] , [86] . Changes in ambient temperature affect interactions between Hsp90 and Hsf1 ( Figure 2 ), and probably affect Hsp90 interactions with Hog1, Mkc1 and Cek1 [49] thereby modulating the activities of these signalling pathways and their inputs to thermal adaptation. Increases in ambient temperature activate Hsf1, thereby inducing the expression of protein chaperones (HSPs) including Hsp90, which promotes thermal adaptation in the shorter term. Hsp90 then down-regulates Hsf1 and modulates Mkc1, Hog1 and Cek1 signalling, which in the longer term influences cell wall architecture ( Figure 11 ), leading to the thermotolerance of C. albicans .
    Figure Legend Snippet: Hsp90 coordinates the activities of multiple signalling pathways that contribute to thermotolerance in C. albicans - a model. Hsf1 activation is required for thermotolerance [42] . Hog1, Mkc1 and Cek1 signalling are also required for thermotolerance ( Figure 3 ), but these MAP kinases are not essential for Hsf1 phosphorylation ( Figure 4 ). Instead, these pathways promote thermotolerance in part via cell wall remodelling [81] , [90] . Hsp90 coordinates much of this activity. Hsf1 ( Figures 1 and 2 ), Hog1, Mkc1 and Cek1 ( Figure 10 ) are all Hsp90 client proteins [62] , [86] . Changes in ambient temperature affect interactions between Hsp90 and Hsf1 ( Figure 2 ), and probably affect Hsp90 interactions with Hog1, Mkc1 and Cek1 [49] thereby modulating the activities of these signalling pathways and their inputs to thermal adaptation. Increases in ambient temperature activate Hsf1, thereby inducing the expression of protein chaperones (HSPs) including Hsp90, which promotes thermal adaptation in the shorter term. Hsp90 then down-regulates Hsf1 and modulates Mkc1, Hog1 and Cek1 signalling, which in the longer term influences cell wall architecture ( Figure 11 ), leading to the thermotolerance of C. albicans .

    Techniques Used: Activation Assay, Activity Assay, Expressing

    Hsp90 depletion affects MAP kinase signalling in the presence and absence of stress. C. albicans wild-type and tetO-HSP90 cells (SN95 and CaLC1411: Table 1 ) were treated with 0 or 20 µg/ml doxycycline for seven hours. Mkc1, Cek1 and Hog1 phosphorylation levels were then assayed by western analysis in unstressed cells or cells treated as follows: (A) 30 minute 30°C–42°C heat shock; (B) 30 minutes with 100 µg/ml Calcofluor White (CFW); (C) 10 minutes with 5 mM H 2 O 2 ; or (D) 12 minutes with 1 M NaCl. Total Mkc1 levels were assayed using Mkc1-6xHis-FLAG tagged cells in SN95 (CaLC681) and tetO-HSP90 (caLC648) cells ( Table 1 ). Hsp90 levels and total kinase levels for Hog1 were also examined by western blotting relative to the internal Act1 loading control.
    Figure Legend Snippet: Hsp90 depletion affects MAP kinase signalling in the presence and absence of stress. C. albicans wild-type and tetO-HSP90 cells (SN95 and CaLC1411: Table 1 ) were treated with 0 or 20 µg/ml doxycycline for seven hours. Mkc1, Cek1 and Hog1 phosphorylation levels were then assayed by western analysis in unstressed cells or cells treated as follows: (A) 30 minute 30°C–42°C heat shock; (B) 30 minutes with 100 µg/ml Calcofluor White (CFW); (C) 10 minutes with 5 mM H 2 O 2 ; or (D) 12 minutes with 1 M NaCl. Total Mkc1 levels were assayed using Mkc1-6xHis-FLAG tagged cells in SN95 (CaLC681) and tetO-HSP90 (caLC648) cells ( Table 1 ). Hsp90 levels and total kinase levels for Hog1 were also examined by western blotting relative to the internal Act1 loading control.

    Techniques Used: Western Blot

    Cross-talk between MAP kinase pathways during heat shock. Differential MAP kinase activation in response to a 30°C–42°C heat shock in MAP kinase mutants. (A) Activation of Hog1, Mkc1 and Cek1 was determined in an mkc1 Δ mutant relative to the internal Act1 control. (B) Activation of Hog1, Mkc1 and Cek1 was assayed in a cek1 Δ mutant relative to the internal Act1 control. (C) Activation of Hog1, Mkc1 and Cek1 was determined in a hog1 Δ mutant relative to the internal Act1 control.
    Figure Legend Snippet: Cross-talk between MAP kinase pathways during heat shock. Differential MAP kinase activation in response to a 30°C–42°C heat shock in MAP kinase mutants. (A) Activation of Hog1, Mkc1 and Cek1 was determined in an mkc1 Δ mutant relative to the internal Act1 control. (B) Activation of Hog1, Mkc1 and Cek1 was assayed in a cek1 Δ mutant relative to the internal Act1 control. (C) Activation of Hog1, Mkc1 and Cek1 was determined in a hog1 Δ mutant relative to the internal Act1 control.

    Techniques Used: Activation Assay, Mutagenesis

    Differential activation profiles of MAP kinases in response to heat shock. (A) Phosphorylation of C. albicans Mkc1, Cek1 and Hog1 during a 30°C–42°C heat shock revealed by western analysis using phospho-specific antibodies. Total kinase levels were monitored using antibodies against total Hog1, FLAG-tagged Mkc1 and TAP-tagged Cek1. Actin served as an internal loading control. (B) Expression of target genes for Mkc1 ( PGA13 ), Cek1 ( PMT4 ) and Hog1 ( RHR2 ) during a 30°C–42°C heat shock, as determined by qRT-PCR of the corresponding transcripts relative to the internal ACT1 mRNA control.
    Figure Legend Snippet: Differential activation profiles of MAP kinases in response to heat shock. (A) Phosphorylation of C. albicans Mkc1, Cek1 and Hog1 during a 30°C–42°C heat shock revealed by western analysis using phospho-specific antibodies. Total kinase levels were monitored using antibodies against total Hog1, FLAG-tagged Mkc1 and TAP-tagged Cek1. Actin served as an internal loading control. (B) Expression of target genes for Mkc1 ( PGA13 ), Cek1 ( PMT4 ) and Hog1 ( RHR2 ) during a 30°C–42°C heat shock, as determined by qRT-PCR of the corresponding transcripts relative to the internal ACT1 mRNA control.

    Techniques Used: Activation Assay, Western Blot, Expressing, Quantitative RT-PCR

    Impact of MAP kinase cross-talk upon stress resistance during thermal adaptation. Cek1 signalling is required for the Calcofluor White resistance of hog1 cells at high temperatures. MIC assays were performed in YPD medium supplemented with different concentrations of NaCl, Calcofluor White or Congo Red. Plates were incubated statically at 25°C, 30°C, 37°C and 42°C for 48 hours. For each strain, optical densities were averaged for duplicate measurements and growth is quantitatively displayed with colour as indicated with the colour bar. Data are representative of three biological replicates. WT, wild type (NGY152: Table 1 ).
    Figure Legend Snippet: Impact of MAP kinase cross-talk upon stress resistance during thermal adaptation. Cek1 signalling is required for the Calcofluor White resistance of hog1 cells at high temperatures. MIC assays were performed in YPD medium supplemented with different concentrations of NaCl, Calcofluor White or Congo Red. Plates were incubated statically at 25°C, 30°C, 37°C and 42°C for 48 hours. For each strain, optical densities were averaged for duplicate measurements and growth is quantitatively displayed with colour as indicated with the colour bar. Data are representative of three biological replicates. WT, wild type (NGY152: Table 1 ).

    Techniques Used: Incubation

    9) Product Images from "Unique Evolution of the UPR Pathway with a Novel bZIP Transcription Factor, Hxl1, for Controlling Pathogenicity of Cryptococcus neoformans"

    Article Title: Unique Evolution of the UPR Pathway with a Novel bZIP Transcription Factor, Hxl1, for Controlling Pathogenicity of Cryptococcus neoformans

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002177

    Functional analyses of C. neoformans HXL1 deletion and complementation. (A) For sensitive test to UPR- and cell wall- stresses, strains were serially diluted and spotted on YPD media with or without exposure to ER stress agents and cell wall disturbing agents (0.075 µg/ml TM, 10 mM DTT, 5 mg/ml CR, 1.5 mg/ml CFW), without (top panel) or with 1 M sorbitol (bottom panel) and incubated at 30°C for 3.5 days. (B) For the thermosensitivity test, strains were spotted on YPD medium and incubated at 35°C or 37°C for 3.5 days. (C) Phosphorylation of Mpk1 induced by ER stress and loss of UPR function. Strains in early exponential phase were treated with TM (5 µg/ml) at 30°C for 2 hr. 30 µg of total protein was analyzed by Western blotting with phospho-p44/42-MAPK antibody (upper panel) for the phosphorylated Mpk1 (Mpk1-P) protein and anti-Hog1 antibody (lower panel) for the Hog1 protein as a loading control. Strains were: wild-type (H99), ire1 (YSB552), ire1+IRE1 complemented (YSB1000), hxl1 (YSB723), hxl1 + HXL1 u complemented (YSB762), ire1 + HXL1 u suppressed (YSB743), ire1 + HXL1 s suppressed (YSB747), mpk 1 (KK3), cna1 (KK1), ras1 (YSB53), and hog1 (YSB64).
    Figure Legend Snippet: Functional analyses of C. neoformans HXL1 deletion and complementation. (A) For sensitive test to UPR- and cell wall- stresses, strains were serially diluted and spotted on YPD media with or without exposure to ER stress agents and cell wall disturbing agents (0.075 µg/ml TM, 10 mM DTT, 5 mg/ml CR, 1.5 mg/ml CFW), without (top panel) or with 1 M sorbitol (bottom panel) and incubated at 30°C for 3.5 days. (B) For the thermosensitivity test, strains were spotted on YPD medium and incubated at 35°C or 37°C for 3.5 days. (C) Phosphorylation of Mpk1 induced by ER stress and loss of UPR function. Strains in early exponential phase were treated with TM (5 µg/ml) at 30°C for 2 hr. 30 µg of total protein was analyzed by Western blotting with phospho-p44/42-MAPK antibody (upper panel) for the phosphorylated Mpk1 (Mpk1-P) protein and anti-Hog1 antibody (lower panel) for the Hog1 protein as a loading control. Strains were: wild-type (H99), ire1 (YSB552), ire1+IRE1 complemented (YSB1000), hxl1 (YSB723), hxl1 + HXL1 u complemented (YSB762), ire1 + HXL1 u suppressed (YSB743), ire1 + HXL1 s suppressed (YSB747), mpk 1 (KK3), cna1 (KK1), ras1 (YSB53), and hog1 (YSB64).

    Techniques Used: Functional Assay, Incubation, Western Blot

    10) Product Images from "Key within-membrane residues and precursor dosage impact the allotopic expression of yeast subunit II of cytochrome c oxidase"

    Article Title: Key within-membrane residues and precursor dosage impact the allotopic expression of yeast subunit II of cytochrome c oxidase

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E18-12-0788

    Substituting residues of properties similar to those of arginine in position 56 of allotopic Cox2 restores cellular respiration to a cox2 -null strain. (A) Tenfold serial dilutions of yeast liquid cultures from strains transformed with the indicated constructs or with an empty vector (plasmid) were plated on fermentable (glucose), respiratory (ethanol + glycerol), and minimal, selective media (-ura or -arg, for verifying the presence of the transforming plasmid or the mitochondrial genome, respectively). (B) Immunoblots decorated with an anti-Cox2 antibody of cytosolic (c; 100-µg) and mitochondrial (m; 50-µg) fractions of the indicated yeast strains. The Cox2 precursor and mature forms of Cox2 are indicated. Antibodies anti-Atp2 and anti-Oxa1 were used to immunodetect the corresponding mitochondrial markers and anti-Hog1 was used for the cytosolic marker. Squared brackets indicate Cox2 variants expressed from plasmids. All the Cox2 variants contain an MTS from Oxa1.
    Figure Legend Snippet: Substituting residues of properties similar to those of arginine in position 56 of allotopic Cox2 restores cellular respiration to a cox2 -null strain. (A) Tenfold serial dilutions of yeast liquid cultures from strains transformed with the indicated constructs or with an empty vector (plasmid) were plated on fermentable (glucose), respiratory (ethanol + glycerol), and minimal, selective media (-ura or -arg, for verifying the presence of the transforming plasmid or the mitochondrial genome, respectively). (B) Immunoblots decorated with an anti-Cox2 antibody of cytosolic (c; 100-µg) and mitochondrial (m; 50-µg) fractions of the indicated yeast strains. The Cox2 precursor and mature forms of Cox2 are indicated. Antibodies anti-Atp2 and anti-Oxa1 were used to immunodetect the corresponding mitochondrial markers and anti-Hog1 was used for the cytosolic marker. Squared brackets indicate Cox2 variants expressed from plasmids. All the Cox2 variants contain an MTS from Oxa1.

    Techniques Used: Transformation Assay, Construct, Plasmid Preparation, Western Blot, Marker

    The allotopically synthesized proteins face the limiting step by aggregating at the mitochondrial periphery. (A) Immunoblots decorated with anti-Cox2 of cytosolic (c; 100-µg) and mitochondrial (m; 50-µg) fractions. Arrows indicate the precursor and mature forms of Cox2. The mitochondrial markers Oxa1 and the cytosolic marker Hog1 were decorated with their corresponding antibodies. (B) Isolated mitochondria (50 µg) were treated with proteinase K (PK) to degrade proteins external to the outer membrane. Parallel samples were preincubated with Triton X-100 to dissolve the membranes and to make all mitochondrial proteins accessible to protease degradation. The asterisk indicates partial degradation of Cox2, possibly due to imperfect mitochondrial preparation. (C) Carbonate extraction separating the membrane extrinsic proteins in the supernatant (S) from the integral membrane proteins in the pellet (P) from yeast expressing the indicated constructs. Atp2 was used as a soluble protein marker and Oxa1 as an integral membrane protein marker. (D) Triton X-100 extraction separating the detergent-solubilized proteins in the supernatant (S) from the (detergent-resistant) aggregated proteins in the pellet (P) from yeast expressing the indicated constructs. Cox3 was used as a solubilized membrane protein marker. Squared brackets indicate Cox2 variants expressed from plasmids. All the Cox2 variants contain an MTS from Oxa1.
    Figure Legend Snippet: The allotopically synthesized proteins face the limiting step by aggregating at the mitochondrial periphery. (A) Immunoblots decorated with anti-Cox2 of cytosolic (c; 100-µg) and mitochondrial (m; 50-µg) fractions. Arrows indicate the precursor and mature forms of Cox2. The mitochondrial markers Oxa1 and the cytosolic marker Hog1 were decorated with their corresponding antibodies. (B) Isolated mitochondria (50 µg) were treated with proteinase K (PK) to degrade proteins external to the outer membrane. Parallel samples were preincubated with Triton X-100 to dissolve the membranes and to make all mitochondrial proteins accessible to protease degradation. The asterisk indicates partial degradation of Cox2, possibly due to imperfect mitochondrial preparation. (C) Carbonate extraction separating the membrane extrinsic proteins in the supernatant (S) from the integral membrane proteins in the pellet (P) from yeast expressing the indicated constructs. Atp2 was used as a soluble protein marker and Oxa1 as an integral membrane protein marker. (D) Triton X-100 extraction separating the detergent-solubilized proteins in the supernatant (S) from the (detergent-resistant) aggregated proteins in the pellet (P) from yeast expressing the indicated constructs. Cox3 was used as a solubilized membrane protein marker. Squared brackets indicate Cox2 variants expressed from plasmids. All the Cox2 variants contain an MTS from Oxa1.

    Techniques Used: Synthesized, Western Blot, Marker, Isolation, Expressing, Construct

    11) Product Images from "Thioredoxin Regulates Multiple Hydrogen Peroxide-Induced Signaling Pathways in Candida albicans ▿"

    Article Title: Thioredoxin Regulates Multiple Hydrogen Peroxide-Induced Signaling Pathways in Candida albicans ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00313-10

    Model depicting the multiple roles of Trx1 in H 2 O 2 signaling in C. albicans . Trx1 acts as a repressor of Cap1 activation and polarized cell growth and as an activator of the Hog1 SAPK. Upon exposure to H 2 O 2 , Trx1 becomes oxidized (ox) by reducing (red) oxidized substrates, such as the 2-Cys peroxiredoxin Tsa1. In the model, oxidation of Trx1 relieves the inhibitory effects of the protein on both Cap1 activation and polarized cell growth. However, in contrast, Trx1 and Tsa1 are both required for the activation of Hog1. One model is that Trx1 function acts to prevent Tsa1 becoming trapped in a form (dimeric) that is unable to activate Hog1, although it is also possible that Trx1 and Tsa1 function independently to regulate H 2 O 2 -induced activation of Hog1. Trx1 clearly has multiple roles in ROS signaling in C. albicans , and consistent with this central role in oxidative-stress responses, trx1 Δ cells display significantly attenuated virulence in a mouse model of disease.
    Figure Legend Snippet: Model depicting the multiple roles of Trx1 in H 2 O 2 signaling in C. albicans . Trx1 acts as a repressor of Cap1 activation and polarized cell growth and as an activator of the Hog1 SAPK. Upon exposure to H 2 O 2 , Trx1 becomes oxidized (ox) by reducing (red) oxidized substrates, such as the 2-Cys peroxiredoxin Tsa1. In the model, oxidation of Trx1 relieves the inhibitory effects of the protein on both Cap1 activation and polarized cell growth. However, in contrast, Trx1 and Tsa1 are both required for the activation of Hog1. One model is that Trx1 function acts to prevent Tsa1 becoming trapped in a form (dimeric) that is unable to activate Hog1, although it is also possible that Trx1 and Tsa1 function independently to regulate H 2 O 2 -induced activation of Hog1. Trx1 clearly has multiple roles in ROS signaling in C. albicans , and consistent with this central role in oxidative-stress responses, trx1 Δ cells display significantly attenuated virulence in a mouse model of disease.

    Techniques Used: Activation Assay

    12) Product Images from "Influence of ylHog1 MAPK kinase on Yarrowia lipolytica stress response and erythritol production"

    Article Title: Influence of ylHog1 MAPK kinase on Yarrowia lipolytica stress response and erythritol production

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33168-6

    Growth of Y. lipolytica strains MK1, yl-hog1Δ and yl-HOG1 after 48 h on YPD agar medium either at the indicated temperature, or supplemented with the indicated stress agents.
    Figure Legend Snippet: Growth of Y. lipolytica strains MK1, yl-hog1Δ and yl-HOG1 after 48 h on YPD agar medium either at the indicated temperature, or supplemented with the indicated stress agents.

    Techniques Used:

    Growth of Y. lipolytica strains MK1, yl-hog1Δ and yl-HOG1 after 48 h on YPD agar medium supplemented with different concentrations of NaCl or sorbitol.
    Figure Legend Snippet: Growth of Y. lipolytica strains MK1, yl-hog1Δ and yl-HOG1 after 48 h on YPD agar medium supplemented with different concentrations of NaCl or sorbitol.

    Techniques Used:

    Growth of Y. lipolytica strains MK1, yl-hog1Δ and yl-HOG1 , in YPD medium supplemented with different concentrations of NaCl: ( a ) without NaCl, ( b ) 0.2 M NaCl, ( c ) 0.4 M NaCl, ( d ) 0.9 M NaCl. OD 600 changes were measured by Bioscreen C. Average was counted from eight repetitions.
    Figure Legend Snippet: Growth of Y. lipolytica strains MK1, yl-hog1Δ and yl-HOG1 , in YPD medium supplemented with different concentrations of NaCl: ( a ) without NaCl, ( b ) 0.2 M NaCl, ( c ) 0.4 M NaCl, ( d ) 0.9 M NaCl. OD 600 changes were measured by Bioscreen C. Average was counted from eight repetitions.

    Techniques Used:

    Production of erythritol by Y. lipolytica strains ( a ) MK1 ( b ) yl-hog1∆ ( c ) yl-HOG1 ( d ) yl-hog1∆ after 8 days of adaptive laboratory evolution. Experiments were performed in triplicate. Some error bars are too small to be visible on the chart.
    Figure Legend Snippet: Production of erythritol by Y. lipolytica strains ( a ) MK1 ( b ) yl-hog1∆ ( c ) yl-HOG1 ( d ) yl-hog1∆ after 8 days of adaptive laboratory evolution. Experiments were performed in triplicate. Some error bars are too small to be visible on the chart.

    Techniques Used:

    13) Product Images from "Redox Regulation, Rather than Stress-Induced Phosphorylation, of a Hog1 Mitogen-Activated Protein Kinase Modulates Its Nitrosative-Stress-Specific Outputs"

    Article Title: Redox Regulation, Rather than Stress-Induced Phosphorylation, of a Hog1 Mitogen-Activated Protein Kinase Modulates Its Nitrosative-Stress-Specific Outputs

    Journal: mBio

    doi: 10.1128/mBio.02229-17

    Redox status of Hog1 following nitrosative stress. (A) The impact of oxidative stress (a 10-min exposure to 5 mM H 2 O 2 ) and nitrosative stress (a 10-min exposure to 2.5 mM DPTA-NONOate) on the redox status of Hog1 in wild-type C. albicans cells (RM1000+Clp20) ( Table S1 ) was analyzed by Western blotting of AMS- and NEM-alkylated extracts with the anti-Hog1 antibody. As shown in the cartoon, proteins were first alkylated with NEM (
    Figure Legend Snippet: Redox status of Hog1 following nitrosative stress. (A) The impact of oxidative stress (a 10-min exposure to 5 mM H 2 O 2 ) and nitrosative stress (a 10-min exposure to 2.5 mM DPTA-NONOate) on the redox status of Hog1 in wild-type C. albicans cells (RM1000+Clp20) ( Table S1 ) was analyzed by Western blotting of AMS- and NEM-alkylated extracts with the anti-Hog1 antibody. As shown in the cartoon, proteins were first alkylated with NEM (

    Techniques Used: Western Blot, Affinity Magnetic Separation

    Hog1 C156S, C161S, and C271S mutations differentially impact the sensitivity to phagocytic killing and virulence of C. albicans . (A) The sensitivity of C. albicans to phagocytic killing was assayed by counting fungal CFU after 2 h of exposure to human PMNs (1:10 ratio of yeast cells to phagocytes). PBS, phosphate-buffered saline control; HOG1/HOG1 , RM1000+CIp20; HOG1/hog1 , Ca2226; hog1/hog1 , JC50; C156S, Ca2222; C161S, Ca2224; C156/161S, Ca2225; C271S, Ca2216 ( Table S1 ). Means and standard deviations from three independent replicate experiments are shown. ns, not significant; ***, P
    Figure Legend Snippet: Hog1 C156S, C161S, and C271S mutations differentially impact the sensitivity to phagocytic killing and virulence of C. albicans . (A) The sensitivity of C. albicans to phagocytic killing was assayed by counting fungal CFU after 2 h of exposure to human PMNs (1:10 ratio of yeast cells to phagocytes). PBS, phosphate-buffered saline control; HOG1/HOG1 , RM1000+CIp20; HOG1/hog1 , Ca2226; hog1/hog1 , JC50; C156S, Ca2222; C161S, Ca2224; C156/161S, Ca2225; C271S, Ca2216 ( Table S1 ). Means and standard deviations from three independent replicate experiments are shown. ns, not significant; ***, P

    Techniques Used:

    Hog1 contributes to the transcriptional response to nitrosative stress. (A) Transcript profiling (RNA sequencing) was performed on three independent replicates cultures of C. albicans HOG1 (RM1000+Clp20) ( Table S1 ) and hog1Δ cells (JC50) exposed to 0 or 2.5 mM DPTA-NONOate for 10 min. In wild-type cells, 321 transcripts displayed statistically significant ( > 2-fold) increases in level in response to the nitrosative stress. Of these, 205 transcripts (64%) were considered to be Hog1-dependent (red nodes) because they were not induced ≥2-fold by nitrosative stress in hog1Δ cells. The remaining 116 transcripts were considered to be Hog1 independent because they were still induced > 2-fold in hog1Δ cells. Using Cytoscape, GO term analysis was performed on these gene subsets, and the outputs were displayed as a gene function network in which the red nodes represent Hog1-dependent genes, the dark grey nodes represent Hog1-independent genes, and the central hubs are colored according to the functional category. For example, blue hubs relate to stress, green hubs relate to metabolism, and the light-gray hub is for genes of unknown function. (B) The levels of intracellular ROS were assayed in C. albicans strains exposed to 0 or 2.5 mM DPTA-NONOate by performing cytometry on DHE-stained cells. Wild type (WT), gray; hog1 Δ, red; cta4 Δ, green; cap1 Δ, yellow. DHE intensity is presented on a log scale, and the mean fluorescent intensity for each cytometry profile is shown. The data shown are representative of three independent experiments. (C) The fold induction of classical nitrosative-stress ( YHB1 ) and oxidative-stress ( TRR1 ) transcripts is shown following exposure of the same C. albicans strains to 2.5 mM DPTA-NONOate for 30 min. Using qRT-PCR, transcript levels were measured relative to the internal ACT1 mRNA control and normalized to the level of that transcript in the absence of the stress. Means and standard deviations are shown for three independent replicate experiments. *, P
    Figure Legend Snippet: Hog1 contributes to the transcriptional response to nitrosative stress. (A) Transcript profiling (RNA sequencing) was performed on three independent replicates cultures of C. albicans HOG1 (RM1000+Clp20) ( Table S1 ) and hog1Δ cells (JC50) exposed to 0 or 2.5 mM DPTA-NONOate for 10 min. In wild-type cells, 321 transcripts displayed statistically significant ( > 2-fold) increases in level in response to the nitrosative stress. Of these, 205 transcripts (64%) were considered to be Hog1-dependent (red nodes) because they were not induced ≥2-fold by nitrosative stress in hog1Δ cells. The remaining 116 transcripts were considered to be Hog1 independent because they were still induced > 2-fold in hog1Δ cells. Using Cytoscape, GO term analysis was performed on these gene subsets, and the outputs were displayed as a gene function network in which the red nodes represent Hog1-dependent genes, the dark grey nodes represent Hog1-independent genes, and the central hubs are colored according to the functional category. For example, blue hubs relate to stress, green hubs relate to metabolism, and the light-gray hub is for genes of unknown function. (B) The levels of intracellular ROS were assayed in C. albicans strains exposed to 0 or 2.5 mM DPTA-NONOate by performing cytometry on DHE-stained cells. Wild type (WT), gray; hog1 Δ, red; cta4 Δ, green; cap1 Δ, yellow. DHE intensity is presented on a log scale, and the mean fluorescent intensity for each cytometry profile is shown. The data shown are representative of three independent experiments. (C) The fold induction of classical nitrosative-stress ( YHB1 ) and oxidative-stress ( TRR1 ) transcripts is shown following exposure of the same C. albicans strains to 2.5 mM DPTA-NONOate for 30 min. Using qRT-PCR, transcript levels were measured relative to the internal ACT1 mRNA control and normalized to the level of that transcript in the absence of the stress. Means and standard deviations are shown for three independent replicate experiments. *, P

    Techniques Used: RNA Sequencing Assay, Functional Assay, Cytometry, Staining, Quantitative RT-PCR

    Hog1 is required for nitrosative-stress resistance. (A) To assay stress resistance, the following C. albicans strains were spotted in 10-fold serial dilutions on plates containing no stress (control), 25 mM succinic acid (SA), 5 mM NaNO 2 , 25 mM succinic acid plus 5 mM NaNO 2 , 0.01 mM NaOH, or 2.5 mM DPTA-NONOate in 0.01 mM NaOH: RM1000+CIp20 ( HOG1/HOG1 ), Ca2226 ( HOG1/hog1 ), JC50 ( hog1/hog1 ), and JC76 ( hog1 AF /hog1 ) (see Table S1 in the supplemental material). All figure panels were derived from the same set of plates. (B) To examine Hog1 phosphorylation following exposure to stress, C. albicans RM1000+CIp20 cells were exposed to stress and extracts were prepared and subjected to Western blotting with a phospho-p38 antibody to detect phosphorylated Hog1 (P-Hog1) and with an anti-Hog1 antibody to detect total Hog1 levels (Hog1) after 10 min of exposure to 1 M NaCl and 10 min of exposure to the DPTA-NONOate carrier NaOH at 0.01 mM; then, expression was detected after exposure to 2.5 mM DPTA-NONOate, 6.0 mM DPTA-NONOate, or 5 mM NaNO 2 at the indicated times (in minutes). All figure panels were derived from the same set of Western blots. (C) Localization of Hog1-YFP in C. albicans JC63 cells exposed for 10 min to no stress (control), 5 mM H 2 O 2 , 2.5 mM DPTA-NONOate, or 5 mM NaNO 2 . Nuclei were counterstained with DAPI. Representative images of cells examined by differential interference contrast (DIC) and fluorescence microscopy (Hog1-YFP and DAPI) are shown.
    Figure Legend Snippet: Hog1 is required for nitrosative-stress resistance. (A) To assay stress resistance, the following C. albicans strains were spotted in 10-fold serial dilutions on plates containing no stress (control), 25 mM succinic acid (SA), 5 mM NaNO 2 , 25 mM succinic acid plus 5 mM NaNO 2 , 0.01 mM NaOH, or 2.5 mM DPTA-NONOate in 0.01 mM NaOH: RM1000+CIp20 ( HOG1/HOG1 ), Ca2226 ( HOG1/hog1 ), JC50 ( hog1/hog1 ), and JC76 ( hog1 AF /hog1 ) (see Table S1 in the supplemental material). All figure panels were derived from the same set of plates. (B) To examine Hog1 phosphorylation following exposure to stress, C. albicans RM1000+CIp20 cells were exposed to stress and extracts were prepared and subjected to Western blotting with a phospho-p38 antibody to detect phosphorylated Hog1 (P-Hog1) and with an anti-Hog1 antibody to detect total Hog1 levels (Hog1) after 10 min of exposure to 1 M NaCl and 10 min of exposure to the DPTA-NONOate carrier NaOH at 0.01 mM; then, expression was detected after exposure to 2.5 mM DPTA-NONOate, 6.0 mM DPTA-NONOate, or 5 mM NaNO 2 at the indicated times (in minutes). All figure panels were derived from the same set of Western blots. (C) Localization of Hog1-YFP in C. albicans JC63 cells exposed for 10 min to no stress (control), 5 mM H 2 O 2 , 2.5 mM DPTA-NONOate, or 5 mM NaNO 2 . Nuclei were counterstained with DAPI. Representative images of cells examined by differential interference contrast (DIC) and fluorescence microscopy (Hog1-YFP and DAPI) are shown.

    Techniques Used: Derivative Assay, Western Blot, Expressing, Fluorescence, Microscopy

    Stress-specific Hog1 outputs are differentially affected by C156, C161, and C271. (A) Impact of C156S, C161S, and C271S mutations upon the redox status of Hog1 following 10 min of exposure to 2.5 mM DPTA-NONOate, as revealed by AMS gels (see the legend to Fig. 3A ). The differences in Hog1 masses between wild-type cells and HOG1 C→S mutants reflect changes in disulfide bond formation in Hog1 in the absence and presence of stress. WT, Ca2226; C156S, Ca2222; C161S, Ca2224; C156/161S, Ca2225; C271S, Ca2216 ( Table S1 ). For the C156/161S columns, the wild-type controls were from the same blot as that showing the lanes with (+) and without (−) DPTA-NONOate and treatment with NEM, DTT, and AMS in Fig. 3A . (B) Impact of C156S, C161S, and C271S mutations upon the nitrosative-, oxidative-, and osmotic-stress sensitivity of C. albicans . Strains were spotted in 10-fold serial dilutions onto plates containing no stress (control), 1 M NaCl, 5 mM H 2 O 2 , 5 mM NaNO 2 , or 2.5 mM DPTA-NONOate. All figure panels for the wild type, null mutant, and C156S and C161S single and double mutants were derived from the same set of plates. The C271 mutant, which displayed no obvious stress phenotype, was compared to the wild-type control in a separate experiment. (C) Impact of C156S, C161S, and C271S mutations upon the phosphorylation dynamics of Hog1 phosphorylation following exposure of cells to 2.5 mM DPTA-NONOate or 5 mM H 2 O 2 . Western blotting was performed on cell extracts prepared at the times indicated (in minutes). Phosphorylated Hog1 (P-Hog1) was detected by probing the blots with phospho-p38 antibody, and for loading controls, the blots were reprobed for total Hog1 and actin. The results are indicative of three independent replicate experiments, and the data for the individual HOG1 C156S and HOG1 C161S mutants are shown in Fig. S4 . (D) Impact of C156S, C161S, and C271S mutations upon the Hog1-mediated induction of nitrosative ( YHB1 )- and oxidative ( TRR1 )-stress genes in response to 5 mM H 2 O 2 or 2.5 mM DPTA-NONOate for 10 min. The strains are as described for panel A. The fold induction of the YHB1 and TRR1 transcripts was measured by qRT-PCR, relative to the internal ACT1 mRNA control, and by normalizing to the levels of these transcripts in the absence of the stress. Means and standard deviations are shown for three independent replicate experiments. *, P
    Figure Legend Snippet: Stress-specific Hog1 outputs are differentially affected by C156, C161, and C271. (A) Impact of C156S, C161S, and C271S mutations upon the redox status of Hog1 following 10 min of exposure to 2.5 mM DPTA-NONOate, as revealed by AMS gels (see the legend to Fig. 3A ). The differences in Hog1 masses between wild-type cells and HOG1 C→S mutants reflect changes in disulfide bond formation in Hog1 in the absence and presence of stress. WT, Ca2226; C156S, Ca2222; C161S, Ca2224; C156/161S, Ca2225; C271S, Ca2216 ( Table S1 ). For the C156/161S columns, the wild-type controls were from the same blot as that showing the lanes with (+) and without (−) DPTA-NONOate and treatment with NEM, DTT, and AMS in Fig. 3A . (B) Impact of C156S, C161S, and C271S mutations upon the nitrosative-, oxidative-, and osmotic-stress sensitivity of C. albicans . Strains were spotted in 10-fold serial dilutions onto plates containing no stress (control), 1 M NaCl, 5 mM H 2 O 2 , 5 mM NaNO 2 , or 2.5 mM DPTA-NONOate. All figure panels for the wild type, null mutant, and C156S and C161S single and double mutants were derived from the same set of plates. The C271 mutant, which displayed no obvious stress phenotype, was compared to the wild-type control in a separate experiment. (C) Impact of C156S, C161S, and C271S mutations upon the phosphorylation dynamics of Hog1 phosphorylation following exposure of cells to 2.5 mM DPTA-NONOate or 5 mM H 2 O 2 . Western blotting was performed on cell extracts prepared at the times indicated (in minutes). Phosphorylated Hog1 (P-Hog1) was detected by probing the blots with phospho-p38 antibody, and for loading controls, the blots were reprobed for total Hog1 and actin. The results are indicative of three independent replicate experiments, and the data for the individual HOG1 C156S and HOG1 C161S mutants are shown in Fig. S4 . (D) Impact of C156S, C161S, and C271S mutations upon the Hog1-mediated induction of nitrosative ( YHB1 )- and oxidative ( TRR1 )-stress genes in response to 5 mM H 2 O 2 or 2.5 mM DPTA-NONOate for 10 min. The strains are as described for panel A. The fold induction of the YHB1 and TRR1 transcripts was measured by qRT-PCR, relative to the internal ACT1 mRNA control, and by normalizing to the levels of these transcripts in the absence of the stress. Means and standard deviations are shown for three independent replicate experiments. *, P

    Techniques Used: Affinity Magnetic Separation, Mutagenesis, Derivative Assay, Western Blot, Quantitative RT-PCR

    14) Product Images from "Blocking two-component signalling enhances Candida albicans virulence and reveals adaptive mechanisms that counteract sustained SAPK activation"

    Article Title: Blocking two-component signalling enhances Candida albicans virulence and reveals adaptive mechanisms that counteract sustained SAPK activation

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006131

    C . albicans cells lacking YPD1 exhibit hyperactivation of Hog1 but are viable. (A) Strategy to control YPD1 expression. One YPD1 allele was deleted, and the remaining allele placed under the control of the E . coli tet operator ( tetO ) in strain THE1 to generate strain tetO-YPD1 (JC1586). THE1 cells express an E . coli tet repressor– S . cerevisiae Hap4 activation domain fusion protein. In the absence of doxycycline (DOX), this fusion protein binds as a dimer to the tet operator resulting in transcriptional activation. However, doxycycline prevents dimerisation of the fusion protein and blocks transcription. (B) Doxycycline treatment inhibits YPD1 expression. Northern analysis of YPD1 and ACT1 (control) transcript levels in tetO-YPD1 cells treated with doxycycline for the indicated times. (C) Repression or deletion of YPD1 results in a slow growth phenotype. Growth analysis of tetO-YPD1 cells, untreated or treated with doxycycline (top panel), and wild-type (Wt, JC21), ypd1Δ (JC2001) and ypd1Δ+YPD1 (JC2002) cells (bottom panel). (D) Repression or deletion of YPD1 results in constitutive phosphorylation of Hog1. Western blot analysis of whole cell extracts isolated from tetO-YPD1 cells following treatment with doxycycline for the indicated times, or from exponentially growing wild-type (Wt), ypd1Δ and ypd1Δ+YPD1 cells. Blots were probed for phosphorylated Hog1 (Hog1-P), stripped and reprobed for total Hog1 (Hog1). (E) Repression or deletion of YPD1 results in high levels of GPD2 and RHR2 expression. RNA was isolated from tetO-YPD1 cells, treated with or without doxycycline for the indicated times, or wild-type and ypd1Δ cells, and analyzed using gene-specific probes with ACT1 as a loading control. (F) Deletion of YPD1 results in increased intracellular glycerol levels. The mean ± SD is shown for 3 biological replicates.
    Figure Legend Snippet: C . albicans cells lacking YPD1 exhibit hyperactivation of Hog1 but are viable. (A) Strategy to control YPD1 expression. One YPD1 allele was deleted, and the remaining allele placed under the control of the E . coli tet operator ( tetO ) in strain THE1 to generate strain tetO-YPD1 (JC1586). THE1 cells express an E . coli tet repressor– S . cerevisiae Hap4 activation domain fusion protein. In the absence of doxycycline (DOX), this fusion protein binds as a dimer to the tet operator resulting in transcriptional activation. However, doxycycline prevents dimerisation of the fusion protein and blocks transcription. (B) Doxycycline treatment inhibits YPD1 expression. Northern analysis of YPD1 and ACT1 (control) transcript levels in tetO-YPD1 cells treated with doxycycline for the indicated times. (C) Repression or deletion of YPD1 results in a slow growth phenotype. Growth analysis of tetO-YPD1 cells, untreated or treated with doxycycline (top panel), and wild-type (Wt, JC21), ypd1Δ (JC2001) and ypd1Δ+YPD1 (JC2002) cells (bottom panel). (D) Repression or deletion of YPD1 results in constitutive phosphorylation of Hog1. Western blot analysis of whole cell extracts isolated from tetO-YPD1 cells following treatment with doxycycline for the indicated times, or from exponentially growing wild-type (Wt), ypd1Δ and ypd1Δ+YPD1 cells. Blots were probed for phosphorylated Hog1 (Hog1-P), stripped and reprobed for total Hog1 (Hog1). (E) Repression or deletion of YPD1 results in high levels of GPD2 and RHR2 expression. RNA was isolated from tetO-YPD1 cells, treated with or without doxycycline for the indicated times, or wild-type and ypd1Δ cells, and analyzed using gene-specific probes with ACT1 as a loading control. (F) Deletion of YPD1 results in increased intracellular glycerol levels. The mean ± SD is shown for 3 biological replicates.

    Techniques Used: Expressing, Activation Assay, Northern Blot, Western Blot, Isolation

    Overview of two component-mediated regulation of the Hog1 SAPK in S . cerevisiae . Under non-stress conditions the Sln1 histidine kinase autophosphorylates, and this phosphate is transferred via the Ypd1 phosphorelay protein to the Ssk1 response regulator protein. Following osmotic stress Sln1 is inactivated, thus halting the phosphorelay which culminates in unphosphorylated Ssk1 which is a potent activator of the Ssk2 MAPKKK. This results in the phosphorylation and activation of the downstream Hog1 SAPK.
    Figure Legend Snippet: Overview of two component-mediated regulation of the Hog1 SAPK in S . cerevisiae . Under non-stress conditions the Sln1 histidine kinase autophosphorylates, and this phosphate is transferred via the Ypd1 phosphorelay protein to the Ssk1 response regulator protein. Following osmotic stress Sln1 is inactivated, thus halting the phosphorelay which culminates in unphosphorylated Ssk1 which is a potent activator of the Ssk2 MAPKKK. This results in the phosphorylation and activation of the downstream Hog1 SAPK.

    Techniques Used: Activation Assay

    The reduction of Hog1 phosphorylation following loss of Ypd1 function can be reversed by stress exposure. (A) Experimental overview. Freshly isolated cells were incubated on rich solid media for 11 days (depicted in grey) and then either re-streaked onto fresh rich media with (+NaCl) or without (-NaCl) 0.3M NaCl (depicted in white) and incubated for a further 2 days, or maintained on the original plate for a further 2 days (13 day). Cells were then cultured overnight in liquid rich media lacking NaCl, and Hog1 phosphorylation and cellular morphology examined. (B) Western blot analysis of both phosphorylated and total Hog1 levels in Wt (JC21) and ypd1Δ (JC2001) cells treated as described in A. (C) Western blot analysis of whole cell extracts isolated from exponentially-growing ypd1Δ cells taken either from rich media plates after the number of days indicated, or after being re-streaked on rich media with NaCl (+NaCl) as described in A. Duplicate blots were probed for phosphorylated Hog1 (Hog1-P) or total Hog1 (Hog1) levels. (D) Micrographs of exponentially-growing Wt and ypd1Δ strains treated as described in A.
    Figure Legend Snippet: The reduction of Hog1 phosphorylation following loss of Ypd1 function can be reversed by stress exposure. (A) Experimental overview. Freshly isolated cells were incubated on rich solid media for 11 days (depicted in grey) and then either re-streaked onto fresh rich media with (+NaCl) or without (-NaCl) 0.3M NaCl (depicted in white) and incubated for a further 2 days, or maintained on the original plate for a further 2 days (13 day). Cells were then cultured overnight in liquid rich media lacking NaCl, and Hog1 phosphorylation and cellular morphology examined. (B) Western blot analysis of both phosphorylated and total Hog1 levels in Wt (JC21) and ypd1Δ (JC2001) cells treated as described in A. (C) Western blot analysis of whole cell extracts isolated from exponentially-growing ypd1Δ cells taken either from rich media plates after the number of days indicated, or after being re-streaked on rich media with NaCl (+NaCl) as described in A. Duplicate blots were probed for phosphorylated Hog1 (Hog1-P) or total Hog1 (Hog1) levels. (D) Micrographs of exponentially-growing Wt and ypd1Δ strains treated as described in A.

    Techniques Used: Isolation, Incubation, Cell Culture, Western Blot

    C . albicans cells adapt to loss of Ypd1 function by inducing negative regulators of Hog1. (A) PTP2 and PTP3 are induced in ypd1Δ cells. Northern blot analysis of PTP2 and PTP3 expression in exponentially growing Wt (JC21 and ypd1Δ (JC2001) cells. ACT1 was used as a loading control. (B) The kinetics of PTP3 and PTP2 are similar to that of Hog1 activation following doxycycline treatment of tetO-YPD1 cells. Northern blot analyses of PTP2 and PTP3 expression, and western blot analysis of Hog1 phosphorylation in tetO-YPD1 cells (JC1586) following treatment with doxycycline for the indicated times. (C) Quantification of PTP3 and PTP2 induction following doxycycline treatment of tetO-YPD1 cells. (D) The tyrosine phosphatase inhibitor, arsenite, further activates Hog1 in ypd1Δ cells. Western blot analysis of whole cell extracts isolated from exponentially growing Wt and ypd1Δ cells treated with 5mM NaAsO 2 for the specified times. Duplicate blots were probed for phosphorylated Hog1 (Hog1-P) or total Hog1 (Hog1) levels. A darker exposure of the Hog1-P blot is included (middle panel) to show the level of Hog1-P observed in Wt cells following arsenite treatment. (E) Hog1 is activated by arsenite in PBS2 DD cells. Western blot analysis of whole cell extracts isolated from exponentially growing PBS2 (JC112), PBS2 AA (JC126) and PBS2 DD (JC124) cells after treatment with 5mM NaAsO 2 for the specified times. Blots were probed for phosphorylated Hog1 (Hog1-P), stripped, and reprobed for total Hog1 (Hog1) levels. (F) Deletion of PTP genes trigger greater activation of Hog1 following repression of YPD1 . Western blot analysis of whole cell extracts isolated from tetO-YPD1 , tetO-YPD1 ptp3Δ (JC2188) and tetO-YPD1 ptp3Δ PTP2/ptp2 (JC2195) cells following treatment with doxycycline for the indicated times. Duplicate blots were probed for phosphorylated Hog1 (Hog1-P) or total Hog1 (Hog1) levels. (G) Deletion of PTP genes impairs cell growth following repression of YPD1 . 10 4 cells, and 10-fold dilutions thereof, of the indicated strains were spotted onto rich media plates plus or minus DOX, and incubated at 30°C for 24h.
    Figure Legend Snippet: C . albicans cells adapt to loss of Ypd1 function by inducing negative regulators of Hog1. (A) PTP2 and PTP3 are induced in ypd1Δ cells. Northern blot analysis of PTP2 and PTP3 expression in exponentially growing Wt (JC21 and ypd1Δ (JC2001) cells. ACT1 was used as a loading control. (B) The kinetics of PTP3 and PTP2 are similar to that of Hog1 activation following doxycycline treatment of tetO-YPD1 cells. Northern blot analyses of PTP2 and PTP3 expression, and western blot analysis of Hog1 phosphorylation in tetO-YPD1 cells (JC1586) following treatment with doxycycline for the indicated times. (C) Quantification of PTP3 and PTP2 induction following doxycycline treatment of tetO-YPD1 cells. (D) The tyrosine phosphatase inhibitor, arsenite, further activates Hog1 in ypd1Δ cells. Western blot analysis of whole cell extracts isolated from exponentially growing Wt and ypd1Δ cells treated with 5mM NaAsO 2 for the specified times. Duplicate blots were probed for phosphorylated Hog1 (Hog1-P) or total Hog1 (Hog1) levels. A darker exposure of the Hog1-P blot is included (middle panel) to show the level of Hog1-P observed in Wt cells following arsenite treatment. (E) Hog1 is activated by arsenite in PBS2 DD cells. Western blot analysis of whole cell extracts isolated from exponentially growing PBS2 (JC112), PBS2 AA (JC126) and PBS2 DD (JC124) cells after treatment with 5mM NaAsO 2 for the specified times. Blots were probed for phosphorylated Hog1 (Hog1-P), stripped, and reprobed for total Hog1 (Hog1) levels. (F) Deletion of PTP genes trigger greater activation of Hog1 following repression of YPD1 . Western blot analysis of whole cell extracts isolated from tetO-YPD1 , tetO-YPD1 ptp3Δ (JC2188) and tetO-YPD1 ptp3Δ PTP2/ptp2 (JC2195) cells following treatment with doxycycline for the indicated times. Duplicate blots were probed for phosphorylated Hog1 (Hog1-P) or total Hog1 (Hog1) levels. (G) Deletion of PTP genes impairs cell growth following repression of YPD1 . 10 4 cells, and 10-fold dilutions thereof, of the indicated strains were spotted onto rich media plates plus or minus DOX, and incubated at 30°C for 24h.

    Techniques Used: Northern Blot, Expressing, Activation Assay, Western Blot, Isolation, Incubation

    C . albicans adapts to long term Ypd1 loss by lowering Hog1 activity. (A) ypd1Δ cells become morphologically similar to wild-type cells over time. Micrographs of exponentially growing Wt (JC21) and ypd1Δ (JC2001) cells taken from rich media plates after 1 or 13 days. (B) ypd1Δ cells gradually accumulate stress phenotypes characteristic of wild-type cells. Approximately 10 4 cells, and 10-fold dilutions thereof, of exponentially growing Wt , hog1Δ (JC50) and ypd1Δ cells taken from rich media plates after 1 or 13 days were spotted onto plates containing; NaAsO 2 (1.5 mM), calcofluor white (CFW 30 μg/ml) and NaCl (0.5 M). Plates were incubated at 30°C for 24 hrs. (C) Hog1 phosphorylation is not sustained in ypd1Δ cells over time. Western blot analysis of whole cell extracts isolated from exponentially growing Wt and ypd1Δ cells taken from rich media plates after the number of days indicated. Blots were probed for phosphorylated Hog1 (Hog1-P), stripped and reprobed for total Hog1 (Hog1). (D) PTP2 , PTP3 , and GPD2 expression is not sustained in ypd1Δ cells. Northern blot analysis of the indicated genes in exponentially growing Wt and ypd1Δ cells taken from plates after the number of days indicated. ACT1 was used as a loading control.
    Figure Legend Snippet: C . albicans adapts to long term Ypd1 loss by lowering Hog1 activity. (A) ypd1Δ cells become morphologically similar to wild-type cells over time. Micrographs of exponentially growing Wt (JC21) and ypd1Δ (JC2001) cells taken from rich media plates after 1 or 13 days. (B) ypd1Δ cells gradually accumulate stress phenotypes characteristic of wild-type cells. Approximately 10 4 cells, and 10-fold dilutions thereof, of exponentially growing Wt , hog1Δ (JC50) and ypd1Δ cells taken from rich media plates after 1 or 13 days were spotted onto plates containing; NaAsO 2 (1.5 mM), calcofluor white (CFW 30 μg/ml) and NaCl (0.5 M). Plates were incubated at 30°C for 24 hrs. (C) Hog1 phosphorylation is not sustained in ypd1Δ cells over time. Western blot analysis of whole cell extracts isolated from exponentially growing Wt and ypd1Δ cells taken from rich media plates after the number of days indicated. Blots were probed for phosphorylated Hog1 (Hog1-P), stripped and reprobed for total Hog1 (Hog1). (D) PTP2 , PTP3 , and GPD2 expression is not sustained in ypd1Δ cells. Northern blot analysis of the indicated genes in exponentially growing Wt and ypd1Δ cells taken from plates after the number of days indicated. ACT1 was used as a loading control.

    Techniques Used: Activity Assay, Incubation, Western Blot, Isolation, Expressing, Northern Blot

    Ypd1 inactivation in C . albicans triggers Hog1 hyperactivation, increased virulence, and in the long term a reduction in Hog1 activity. Model depicting outcomes following YPD1 loss in C . albicans . Loss of Ypd1 results in the accumulation of the unphosphorylated Ssk1 response regulator, which drives the activation of Hog1 under non-stressed conditions. The levels of Hog1 phosphorylation are modulated by the induction of the negative regulators Ptp2 and Ptp3 which allows cells to adapt and survive Ypd1 loss. Loss of Ypd1 function during infection increases the virulence of C . albicans , by possibly enhancing Hog1 activity promoting stress resistance and/or filamentation. Furthermore, C . albicans adapts to long-term activation of Hog1 by reducing the levels of the phosphorylated Hog1 kinase. This adaptation process prevents phenotypes associated with sustained SAPK activation and ypd1Δ cells now phenotypically resemble wild-type cells. Notably, however, this adaptation mechanism to circumvent Hog1 phosphorylation can be over-ridden following transient stress exposure and thus sustained Hog1 activation is restored.
    Figure Legend Snippet: Ypd1 inactivation in C . albicans triggers Hog1 hyperactivation, increased virulence, and in the long term a reduction in Hog1 activity. Model depicting outcomes following YPD1 loss in C . albicans . Loss of Ypd1 results in the accumulation of the unphosphorylated Ssk1 response regulator, which drives the activation of Hog1 under non-stressed conditions. The levels of Hog1 phosphorylation are modulated by the induction of the negative regulators Ptp2 and Ptp3 which allows cells to adapt and survive Ypd1 loss. Loss of Ypd1 function during infection increases the virulence of C . albicans , by possibly enhancing Hog1 activity promoting stress resistance and/or filamentation. Furthermore, C . albicans adapts to long-term activation of Hog1 by reducing the levels of the phosphorylated Hog1 kinase. This adaptation process prevents phenotypes associated with sustained SAPK activation and ypd1Δ cells now phenotypically resemble wild-type cells. Notably, however, this adaptation mechanism to circumvent Hog1 phosphorylation can be over-ridden following transient stress exposure and thus sustained Hog1 activation is restored.

    Techniques Used: Activity Assay, Activation Assay, Infection

    Phenotypes associated with loss of Ypd1 are dependent on Hog1 and Ssk1. (A) Repression or deletion of YPD1 triggers flocculation and a swollen pseudohyphal filamentous phenotype. Micrographs of Wt , ypd1Δ , and tetO-YPD1 cells plus or minus doxycycline (DOX) grown overnight in rich media. Images of culture tubes demonstrate the rapid sedimentation rate of cells lacking YPD1 . (B) Repression or deletion of YPD1 results in pleiotropic stress phenotypes. 10 4 cells, and 10-fold dilutions thereof, of exponentially growing tetO-YPD1 cells, or wild-type ( Wt ), ypd1Δ and ypd1Δ+YPD1 cells, were spotted onto rich media plates (plus or minus DOX for tetO-YPD1 cells) containing NaCl (1.0 M), calcofluor white (CFW, 30 μg/ml), NaAsO 2 (1.5 mM) and t -BOOH (2 mM), and incubated at 30°C for 24h. (C) The morphological defects exhibited by ypd1Δ cells are dependent on Hog1 and Ssk1. Micrographs of wild-type ( Wt ), ypd1Δ , hog1Δ (JC50), ssk1Δ (JC1552), hog1Δ ypd1Δ (JC1475) hog1Δypd1Δ + HOG1 (JC1478), ssk1Δ ypd1Δ (JC1683), and ssk1Δ ypd1Δ + SSK1 (JC1704) cells. (D) The high glycerol levels in ypd1Δ cells are dependent on Hog1. The mean ± SD is shown for 3 biological replicates. (E) The stress phenotypes exhibited by ypd1Δ cells are dependent on Hog1 and Ssk1. Exponentially growing strains were spotted onto rich media plates containing the additives detailed in B above, and incubated at 30°C for 24h. (F) The sustained Hog1 activation in ypd1Δ cells is dependent on Ssk1. Western blots depicting basal levels of Hog1 phosphorylation in the indicated strains. Blots were probed for phosphorylated Hog1 (Hog1-P), stripped and reprobed for total Hog1 (Hog1).
    Figure Legend Snippet: Phenotypes associated with loss of Ypd1 are dependent on Hog1 and Ssk1. (A) Repression or deletion of YPD1 triggers flocculation and a swollen pseudohyphal filamentous phenotype. Micrographs of Wt , ypd1Δ , and tetO-YPD1 cells plus or minus doxycycline (DOX) grown overnight in rich media. Images of culture tubes demonstrate the rapid sedimentation rate of cells lacking YPD1 . (B) Repression or deletion of YPD1 results in pleiotropic stress phenotypes. 10 4 cells, and 10-fold dilutions thereof, of exponentially growing tetO-YPD1 cells, or wild-type ( Wt ), ypd1Δ and ypd1Δ+YPD1 cells, were spotted onto rich media plates (plus or minus DOX for tetO-YPD1 cells) containing NaCl (1.0 M), calcofluor white (CFW, 30 μg/ml), NaAsO 2 (1.5 mM) and t -BOOH (2 mM), and incubated at 30°C for 24h. (C) The morphological defects exhibited by ypd1Δ cells are dependent on Hog1 and Ssk1. Micrographs of wild-type ( Wt ), ypd1Δ , hog1Δ (JC50), ssk1Δ (JC1552), hog1Δ ypd1Δ (JC1475) hog1Δypd1Δ + HOG1 (JC1478), ssk1Δ ypd1Δ (JC1683), and ssk1Δ ypd1Δ + SSK1 (JC1704) cells. (D) The high glycerol levels in ypd1Δ cells are dependent on Hog1. The mean ± SD is shown for 3 biological replicates. (E) The stress phenotypes exhibited by ypd1Δ cells are dependent on Hog1 and Ssk1. Exponentially growing strains were spotted onto rich media plates containing the additives detailed in B above, and incubated at 30°C for 24h. (F) The sustained Hog1 activation in ypd1Δ cells is dependent on Ssk1. Western blots depicting basal levels of Hog1 phosphorylation in the indicated strains. Blots were probed for phosphorylated Hog1 (Hog1-P), stripped and reprobed for total Hog1 (Hog1).

    Techniques Used: Flocculation, Sedimentation, Incubation, Activation Assay, Western Blot

    15) Product Images from "Influence of ylHog1 MAPK kinase on Yarrowia lipolytica stress response and erythritol production"

    Article Title: Influence of ylHog1 MAPK kinase on Yarrowia lipolytica stress response and erythritol production

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33168-6

    Growth of Y. lipolytica strains MK1, yl-hog1Δ and yl-HOG1 after 48 h on YPD agar medium either at the indicated temperature, or supplemented with the indicated stress agents.
    Figure Legend Snippet: Growth of Y. lipolytica strains MK1, yl-hog1Δ and yl-HOG1 after 48 h on YPD agar medium either at the indicated temperature, or supplemented with the indicated stress agents.

    Techniques Used:

    Growth of Y. lipolytica strains MK1, yl-hog1Δ and yl-HOG1 after 48 h on YPD agar medium supplemented with different concentrations of NaCl or sorbitol.
    Figure Legend Snippet: Growth of Y. lipolytica strains MK1, yl-hog1Δ and yl-HOG1 after 48 h on YPD agar medium supplemented with different concentrations of NaCl or sorbitol.

    Techniques Used:

    Production of erythritol by Y. lipolytica strains ( a ) MK1 ( b ) yl-hog1∆ ( c ) yl-HOG1 ( d ) yl-hog1∆ after 8 days of adaptive laboratory evolution. Experiments were performed in triplicate. Some error bars are too small to be visible on the chart.
    Figure Legend Snippet: Production of erythritol by Y. lipolytica strains ( a ) MK1 ( b ) yl-hog1∆ ( c ) yl-HOG1 ( d ) yl-hog1∆ after 8 days of adaptive laboratory evolution. Experiments were performed in triplicate. Some error bars are too small to be visible on the chart.

    Techniques Used:

    Growth of Y. lipolytica strains MK1, yl-hog1Δ and yl-HOG1 , in YPD medium supplemented with different concentrations of NaCl: ( a ) without NaCl, ( b ) 0.2 M NaCl, ( c ) 0.4 M NaCl, ( d ) 0.9 M NaCl. OD 600 changes were measured by Bioscreen C. Average was counted from eight repetitions.
    Figure Legend Snippet: Growth of Y. lipolytica strains MK1, yl-hog1Δ and yl-HOG1 , in YPD medium supplemented with different concentrations of NaCl: ( a ) without NaCl, ( b ) 0.2 M NaCl, ( c ) 0.4 M NaCl, ( d ) 0.9 M NaCl. OD 600 changes were measured by Bioscreen C. Average was counted from eight repetitions.

    Techniques Used:

    Western blot analysis of whole cell extracts isolated from Y. lipolytica wild type (MK1), yl-hog1∆ and yl-HOG1 cells after treatment with 1 M NaCl for the specified times. The active form of protein (Hog1-P) was detected using an anti-phospho p38 antibody (upper panel). Total levels of yl-Hog1 protein were determined by probing the blot with an anti-Hog1 antibody (lower panel). The full-length blots are presented in Supplementary Fig. 1 .
    Figure Legend Snippet: Western blot analysis of whole cell extracts isolated from Y. lipolytica wild type (MK1), yl-hog1∆ and yl-HOG1 cells after treatment with 1 M NaCl for the specified times. The active form of protein (Hog1-P) was detected using an anti-phospho p38 antibody (upper panel). Total levels of yl-Hog1 protein were determined by probing the blot with an anti-Hog1 antibody (lower panel). The full-length blots are presented in Supplementary Fig. 1 .

    Techniques Used: Western Blot, Isolation

    MAPK pathway leading to activation of Hog1 in S. cerevisiae .
    Figure Legend Snippet: MAPK pathway leading to activation of Hog1 in S. cerevisiae .

    Techniques Used: Activation Assay

    16) Product Images from "Stress-induced nuclear accumulation is dispensable for Hog1-dependent gene expression and virulence in a fungal pathogen"

    Article Title: Stress-induced nuclear accumulation is dispensable for Hog1-dependent gene expression and virulence in a fungal pathogen

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14756-4

    Impact of Hog1 localisation on signal fidelity. ( A ) Cek1 and Mkc1 phosphorylation. Western blot analysis of whole cell extracts from Hog1-GFP, Hog1-GFP-NLS, Hog1-GFP-CaaX, and hog1Δ cells, following 0 and 10 min exposure to 1 M sorbitol or 5 mM H 2 O 2 . Blots were probed for phosphorylated Mkc1 (Mkc1-P) and Cek1 (Cek1-P), stripped and reprobed for tubulin as a loading control. Duplicate blots were also probed for phosphorylated Hog1, stripped and reprobed for total Hog1 (Hog1). Cropped images are shown and full-length blots/gels are presented in Supplementary Figure 4 . ( B ) Stress resistance. Dilutions of mid-exponential C . albicans cultures were spotted onto YPD plates containing no stress (ns) or increasing amounts of calcofluor white (CFW: 20, 30 µg/ml) or caffeine (10 mM) and photographed after 48 h growth at 30 °C. ( D ) Mkc1 phosphorylation in response to caffeine. Blots were processed as described in ( A ) above and full-length blots/gels are presented in Supplementary Figure 4 .
    Figure Legend Snippet: Impact of Hog1 localisation on signal fidelity. ( A ) Cek1 and Mkc1 phosphorylation. Western blot analysis of whole cell extracts from Hog1-GFP, Hog1-GFP-NLS, Hog1-GFP-CaaX, and hog1Δ cells, following 0 and 10 min exposure to 1 M sorbitol or 5 mM H 2 O 2 . Blots were probed for phosphorylated Mkc1 (Mkc1-P) and Cek1 (Cek1-P), stripped and reprobed for tubulin as a loading control. Duplicate blots were also probed for phosphorylated Hog1, stripped and reprobed for total Hog1 (Hog1). Cropped images are shown and full-length blots/gels are presented in Supplementary Figure 4 . ( B ) Stress resistance. Dilutions of mid-exponential C . albicans cultures were spotted onto YPD plates containing no stress (ns) or increasing amounts of calcofluor white (CFW: 20, 30 µg/ml) or caffeine (10 mM) and photographed after 48 h growth at 30 °C. ( D ) Mkc1 phosphorylation in response to caffeine. Blots were processed as described in ( A ) above and full-length blots/gels are presented in Supplementary Figure 4 .

    Techniques Used: Western Blot

    Impact of Hog1 localisation on C . albicans virulence. ( A ) Galleria mellonella model of systemic infection. 5 × 10 5 cells of Hog1-GFP, Hog1-GFP-CaaX, Hog1-GFP-NLS, or hog1Δ strains were injected into the hemocoel at the last left pro-leg of 20 Galleria larvae. Sterile PBS was injected into control larvae. Survival was monitored for 3 days at 37 °C and presented in a Kaplan-Meier plot and analysed using log rank tests. ( B ) Mouse model of infection . Kidney fungal burden measurements, percentage weight loss, and outcome score measurements of mice (n = 10) infected with Hog1-GFP, Hog1-GFP-CaaX, Hog1-GFP-NLS, or hog1Δ strains (4.0 × 10 4 cells). Differences were tested by Kruskal-Wallis statistical analysis; ***p
    Figure Legend Snippet: Impact of Hog1 localisation on C . albicans virulence. ( A ) Galleria mellonella model of systemic infection. 5 × 10 5 cells of Hog1-GFP, Hog1-GFP-CaaX, Hog1-GFP-NLS, or hog1Δ strains were injected into the hemocoel at the last left pro-leg of 20 Galleria larvae. Sterile PBS was injected into control larvae. Survival was monitored for 3 days at 37 °C and presented in a Kaplan-Meier plot and analysed using log rank tests. ( B ) Mouse model of infection . Kidney fungal burden measurements, percentage weight loss, and outcome score measurements of mice (n = 10) infected with Hog1-GFP, Hog1-GFP-CaaX, Hog1-GFP-NLS, or hog1Δ strains (4.0 × 10 4 cells). Differences were tested by Kruskal-Wallis statistical analysis; ***p

    Techniques Used: Infection, Injection, Mouse Assay

    Manipulating the cellular localisation of Hog1. ( A ) Schematic depiction of Hog1-GFP chimeras. ( B ) Western blot analysis of whole cell extracts from wild-type, hog1Δ , Hog1-GFP, Hog1-GFP-NLS and Hog1-GFP-CaaX cells, 0 and 10 min after treatment with 5 mM H 2 O 2 or 1 M NaCl . Blots were probed for Hog1. ( C ) Confocal microscopy of cells expressing Hog1-GFP, Hog1-GFP-NLS, and Hog1-GFP-CaaX constructs, 0 and 10 min after treatment with 5 mM H 2 O 2 or 1 M NaCl. ( D ) Quantification of Hog1-GFP nuclear accumulation. Quantification was performed using Volocity 6.1.1 software and the percentage of nuclear Hog1 (mean ± SEM) relative to that seen in non-stressed cells expressing wild-type Hog1-GFP is shown (n > 10 individual cells). The data were analysed statistically using one-way ANOVA: ns, not significant; *p
    Figure Legend Snippet: Manipulating the cellular localisation of Hog1. ( A ) Schematic depiction of Hog1-GFP chimeras. ( B ) Western blot analysis of whole cell extracts from wild-type, hog1Δ , Hog1-GFP, Hog1-GFP-NLS and Hog1-GFP-CaaX cells, 0 and 10 min after treatment with 5 mM H 2 O 2 or 1 M NaCl . Blots were probed for Hog1. ( C ) Confocal microscopy of cells expressing Hog1-GFP, Hog1-GFP-NLS, and Hog1-GFP-CaaX constructs, 0 and 10 min after treatment with 5 mM H 2 O 2 or 1 M NaCl. ( D ) Quantification of Hog1-GFP nuclear accumulation. Quantification was performed using Volocity 6.1.1 software and the percentage of nuclear Hog1 (mean ± SEM) relative to that seen in non-stressed cells expressing wild-type Hog1-GFP is shown (n > 10 individual cells). The data were analysed statistically using one-way ANOVA: ns, not significant; *p

    Techniques Used: Western Blot, Confocal Microscopy, Expressing, Construct, Software

    Impact of Hog1 localisation on Hog1-dependent phenotypes. ( A ) Stress resistance. Dilutions of mid-exponential C . albicans cultures were spotted onto YPD plates containing no stress (ns) or increasing amounts of NaCl (0.5, 1.0 M), H 2 O 2 (3.0, 4.0 mM) or t-BOOH (1.5, 2.0 mM) and photographed after 48 h growth at 30 °C. ( B ) Hog1 phosphorylation. Western blot analysis of whole cell extracts from Hog1-GFP, Hog1-GFP-NLS and Hog1-GFP-CaaX cells, following treatment with either 1 M NaCl, 5 mM H 2 O 2 , or 2 mM t-BOOH for the indicated times. Blots were probed for phosphorylated Hog1 (Hog1-P), stripped and reprobed for total Hog1 (Hog1). Cropped images are shown and full-length blots/gels are presented in Supplementary Figure 4 . ( C ) Gene-induction. RNA was isolated from the above cells following treatment with 0.3 M NaCl for the indicated times, and analyzed using gene-specific probes with ACT1 as a loading control. The fold-induction of gene expression was quantified relative to that in Hog1-GFP cells before stress and the data are presented as mean +/− SE of two independent experiments. ( D ) Repression of hyphal elongation. Overnight cultures were diluted at 1:100 into YPD medium at 25 or 30 °C and incubated for 3.5 h, or into prewarmed YPD medium at 37 °C for 30 min and then transferred to 30 °C for 3 h for cell morphology analysis.
    Figure Legend Snippet: Impact of Hog1 localisation on Hog1-dependent phenotypes. ( A ) Stress resistance. Dilutions of mid-exponential C . albicans cultures were spotted onto YPD plates containing no stress (ns) or increasing amounts of NaCl (0.5, 1.0 M), H 2 O 2 (3.0, 4.0 mM) or t-BOOH (1.5, 2.0 mM) and photographed after 48 h growth at 30 °C. ( B ) Hog1 phosphorylation. Western blot analysis of whole cell extracts from Hog1-GFP, Hog1-GFP-NLS and Hog1-GFP-CaaX cells, following treatment with either 1 M NaCl, 5 mM H 2 O 2 , or 2 mM t-BOOH for the indicated times. Blots were probed for phosphorylated Hog1 (Hog1-P), stripped and reprobed for total Hog1 (Hog1). Cropped images are shown and full-length blots/gels are presented in Supplementary Figure 4 . ( C ) Gene-induction. RNA was isolated from the above cells following treatment with 0.3 M NaCl for the indicated times, and analyzed using gene-specific probes with ACT1 as a loading control. The fold-induction of gene expression was quantified relative to that in Hog1-GFP cells before stress and the data are presented as mean +/− SE of two independent experiments. ( D ) Repression of hyphal elongation. Overnight cultures were diluted at 1:100 into YPD medium at 25 or 30 °C and incubated for 3.5 h, or into prewarmed YPD medium at 37 °C for 30 min and then transferred to 30 °C for 3 h for cell morphology analysis.

    Techniques Used: Western Blot, Isolation, Expressing, Incubation

    17) Product Images from "Hog1 Regulates Stress Tolerance and Virulence in the Emerging Fungal Pathogen Candida auris"

    Article Title: Hog1 Regulates Stress Tolerance and Virulence in the Emerging Fungal Pathogen Candida auris

    Journal: mSphere

    doi: 10.1128/mSphere.00506-18

    Sequence comparisons of fungal HOG1 orthologues. (A) Multiple-sequence alignment of the indicated Hog1 sequences using Clustal Omega and visualized using Jalview. The top graph shows the sequence conservation: columns are highlighted with the same color where there is conservation across the compared sequences. The graph at the bottom tracks the conservation with identical amino acid sequences highlighted in yellow. C. au ; C. auris , C. al ; C. albicans , C. d ; C. dubliniensis ; S. p ; S. pombe , S. c ; S. cerevisiae , C. g ; C. glabrata . (B) Sequence alignment of the C-terminal Hog1 sequences from C. auris , S. cerevisiae , and C. glabrata . (C) Hog1 mobility. Western blot depicting the size of Hog1 orthologues from the indicated fungal species. The predicted size of each orthologue is shown (kDa).
    Figure Legend Snippet: Sequence comparisons of fungal HOG1 orthologues. (A) Multiple-sequence alignment of the indicated Hog1 sequences using Clustal Omega and visualized using Jalview. The top graph shows the sequence conservation: columns are highlighted with the same color where there is conservation across the compared sequences. The graph at the bottom tracks the conservation with identical amino acid sequences highlighted in yellow. C. au ; C. auris , C. al ; C. albicans , C. d ; C. dubliniensis ; S. p ; S. pombe , S. c ; S. cerevisiae , C. g ; C. glabrata . (B) Sequence alignment of the C-terminal Hog1 sequences from C. auris , S. cerevisiae , and C. glabrata . (C) Hog1 mobility. Western blot depicting the size of Hog1 orthologues from the indicated fungal species. The predicted size of each orthologue is shown (kDa).

    Techniques Used: Sequencing, Western Blot

    Construction and analysis of C. auris hog1 Δ cells. (A) Schematic diagram of the strategy used to delete HOG1 . (B) Western blotting of potential hog1 Δ strains, identified by PCR genotyping, confirmed the deletion of Hog1. Western blot analysis of lysates prepared from the indicated strains and probed with an anti-Hog1 antibody. *, nonspecific band present in all extracts.(C) Deletion of HOG1 impacts C. auris cell morphology. DIC images of exponentially growing wild-type and hog1 Δ C. auris strains. (D) C. auris cells lacking HOG1 aggregate. Micrographs of wild-type and hog1 Δ strains grown overnight in YPD medium. Images of culture tubes demonstrate the rapid sedimentation of cells lacking HOG1 . (E) Deletion of HOG1 impacts resistance to cell-wall-damaging agents. Exponentially growing strains were spotted onto rich medium plates containing the indicated additives and incubated at 30°C for 24h. (F) C. auris hog1 Δ cells exhibit more exposed chitin.
    Figure Legend Snippet: Construction and analysis of C. auris hog1 Δ cells. (A) Schematic diagram of the strategy used to delete HOG1 . (B) Western blotting of potential hog1 Δ strains, identified by PCR genotyping, confirmed the deletion of Hog1. Western blot analysis of lysates prepared from the indicated strains and probed with an anti-Hog1 antibody. *, nonspecific band present in all extracts.(C) Deletion of HOG1 impacts C. auris cell morphology. DIC images of exponentially growing wild-type and hog1 Δ C. auris strains. (D) C. auris cells lacking HOG1 aggregate. Micrographs of wild-type and hog1 Δ strains grown overnight in YPD medium. Images of culture tubes demonstrate the rapid sedimentation of cells lacking HOG1 . (E) Deletion of HOG1 impacts resistance to cell-wall-damaging agents. Exponentially growing strains were spotted onto rich medium plates containing the indicated additives and incubated at 30°C for 24h. (F) C. auris hog1 Δ cells exhibit more exposed chitin.

    Techniques Used: Western Blot, Polymerase Chain Reaction, Sedimentation, Incubation

    C. elegans model of infection. (A) C. auris displays comparable virulence to C. albicans in C. elegans . (B) Deletion of Hog1 attenuates C. auris virulence in C. elegans . In both experiments, nematodes were infected with the indicated strains and the survival was monitored daily. These data are from a single experiment; two further independent biological replicates are shown in Fig. S4 in the supplemental material.
    Figure Legend Snippet: C. elegans model of infection. (A) C. auris displays comparable virulence to C. albicans in C. elegans . (B) Deletion of Hog1 attenuates C. auris virulence in C. elegans . In both experiments, nematodes were infected with the indicated strains and the survival was monitored daily. These data are from a single experiment; two further independent biological replicates are shown in Fig. S4 in the supplemental material.

    Techniques Used: Infection

    Stress-protective roles of C. auris Hog1. (A) Hog1 is required for resistance to diverse stresses. Exponentially growing cells were spotted in serial dilutions onto YPD agar plates containing the indicated additives and incubated for 24 or 48 h at 30°C. (B) Hog1 is activated in response to diverse stresses. Western blots depicting Hog1 phosphorylation in response to the indicated stresses. Blots were probed for phosphorylated Hog1 (Hog1-P), stripped, and reprobed for total Hog1 (Hog1).
    Figure Legend Snippet: Stress-protective roles of C. auris Hog1. (A) Hog1 is required for resistance to diverse stresses. Exponentially growing cells were spotted in serial dilutions onto YPD agar plates containing the indicated additives and incubated for 24 or 48 h at 30°C. (B) Hog1 is activated in response to diverse stresses. Western blots depicting Hog1 phosphorylation in response to the indicated stresses. Blots were probed for phosphorylated Hog1 (Hog1-P), stripped, and reprobed for total Hog1 (Hog1).

    Techniques Used: Incubation, Western Blot

    18) Product Images from "A Single MAPKKK Regulates the Hog1 MAPK Pathway in the Pathogenic Fungus Candida albicans"

    Article Title: A Single MAPKKK Regulates the Hog1 MAPK Pathway in the Pathogenic Fungus Candida albicans

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E07-06-0581

    ssk2Δ and hog1 Δ cells display similar phenotypes. (A) Approximately 10 3 cells, and 10-fold dilutions thereof, of exponentially growing wild-type ( Wt -BWP17), hog1 Δ (JC47), ssk2 Δ (JC482), ssk2 Δ+ SSK2 (JC620), ste11
    Figure Legend Snippet: ssk2Δ and hog1 Δ cells display similar phenotypes. (A) Approximately 10 3 cells, and 10-fold dilutions thereof, of exponentially growing wild-type ( Wt -BWP17), hog1 Δ (JC47), ssk2 Δ (JC482), ssk2 Δ+ SSK2 (JC620), ste11

    Techniques Used:

    CaSsk2 is not essential for the interaction of Hog1 with Pbs2. Coprecipitation experiments to examine the association of Hog1 with Pbs2 in wild-type and ssk2 Δ cells. Extracts were prepared from mid-log cultures of Wt (BWP17) and ssk2 Δ
    Figure Legend Snippet: CaSsk2 is not essential for the interaction of Hog1 with Pbs2. Coprecipitation experiments to examine the association of Hog1 with Pbs2 in wild-type and ssk2 Δ cells. Extracts were prepared from mid-log cultures of Wt (BWP17) and ssk2 Δ

    Techniques Used:

    CaSsk2 is required for the activation of Hog1-dependent gene expression. (A) Northern blot analysis of RNA isolated from mid-log cultures of Wt (BWP17), hog1 Δ (JC47), and ssk2 Δ (JC482) cells treated with either 5 mM H 2 O 2 or 0.3 M NaCl
    Figure Legend Snippet: CaSsk2 is required for the activation of Hog1-dependent gene expression. (A) Northern blot analysis of RNA isolated from mid-log cultures of Wt (BWP17), hog1 Δ (JC47), and ssk2 Δ (JC482) cells treated with either 5 mM H 2 O 2 or 0.3 M NaCl

    Techniques Used: Activation Assay, Expressing, Northern Blot, Isolation

    CaSsk2 but not CaSte11 is required for Hog1 phosphorylation. (A) Western blot analysis of whole cell extracts isolated from wild-type ( Wt -BWP17), ssk2 Δ (JC482), ssk2 Δ+ SSK2 (JC620), and ste11 Δ (JC524) cells after treatment with
    Figure Legend Snippet: CaSsk2 but not CaSte11 is required for Hog1 phosphorylation. (A) Western blot analysis of whole cell extracts isolated from wild-type ( Wt -BWP17), ssk2 Δ (JC482), ssk2 Δ+ SSK2 (JC620), and ste11 Δ (JC524) cells after treatment with

    Techniques Used: Western Blot, Isolation

    CaSsk2 but not CaSte11 is required for Hog1 nuclear localization. The localization of YFP-tagged Hog1 was determined by fluorescence microscopy in wild-type ( Wt -JC54), ssk2 Δ (JC522), and ste11 Δ (JC545) cells before stress and after treatment
    Figure Legend Snippet: CaSsk2 but not CaSte11 is required for Hog1 nuclear localization. The localization of YFP-tagged Hog1 was determined by fluorescence microscopy in wild-type ( Wt -JC54), ssk2 Δ (JC522), and ste11 Δ (JC545) cells before stress and after treatment

    Techniques Used: Fluorescence, Microscopy

    Model depicting the relay of osmotic and oxidative stress signals to the Hog1 MAPK in C. albicans
    Figure Legend Snippet: Model depicting the relay of osmotic and oxidative stress signals to the Hog1 MAPK in C. albicans

    Techniques Used:

    19) Product Images from "Functions of Candida albicans cell wall glycosidases Dfg5p and Dcw1p in biofilm formation and HOG MAPK pathway"

    Article Title: Functions of Candida albicans cell wall glycosidases Dfg5p and Dcw1p in biofilm formation and HOG MAPK pathway

    Journal: PeerJ

    doi: 10.7717/peerj.5685

    DFG5 and DCW1 are required for basal Hog1 levels. Western blot analysis was performed using anti-Hog1 antibody to detect whole Hog1 (non-phosphorylated) under (A) non-stress and (B) osmotic stress conditions.
    Figure Legend Snippet: DFG5 and DCW1 are required for basal Hog1 levels. Western blot analysis was performed using anti-Hog1 antibody to detect whole Hog1 (non-phosphorylated) under (A) non-stress and (B) osmotic stress conditions.

    Techniques Used: Western Blot

    Dfg5p and Dcw1 functional role in regulating hyphal morphogenesis and HOG MAPK pathway. The ligands and downstream components involved in DFG5/DCW1 dependent hyphal regulation and Hog1 signaling as well as the cell wall protein substrates for Dfg5p and Dcw1p are to be determined.
    Figure Legend Snippet: Dfg5p and Dcw1 functional role in regulating hyphal morphogenesis and HOG MAPK pathway. The ligands and downstream components involved in DFG5/DCW1 dependent hyphal regulation and Hog1 signaling as well as the cell wall protein substrates for Dfg5p and Dcw1p are to be determined.

    Techniques Used: Functional Assay

    DFG5 and DCW1 heterologous mutations affect Hog1 phosphorylation. Phospho Hog1 and whole Hog1 Western blots. Coomassie staining of the blot was performed loading controls, prior to antibody detection.
    Figure Legend Snippet: DFG5 and DCW1 heterologous mutations affect Hog1 phosphorylation. Phospho Hog1 and whole Hog1 Western blots. Coomassie staining of the blot was performed loading controls, prior to antibody detection.

    Techniques Used: Western Blot, Staining

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    Article Title: The coordinate actions of calcineurin and Hog1 mediate the stress response through multiple nodes of the cell cycle network
    Article Snippet: .. Western blotting was performed with antibodies against Hog1 (sc-6815 or sc-165978, Santa Cruz Biotechnology), phosphorylated Hog1/p38 (anti-Phospho-p38 MAPK, 9211L, Cell Signaling Technology), PSTAIRE (sc-53, Santa Cruz Biotechnology or P7962, Sigma), G6PDH (A9521, Sigma), V5 (R96025, Invitrogen), FLAG (clone M2, F1804, Sigma), MYC (clone 9E10, M5546, Sigma), Y19-phosphorylated Cdk1 (anti-Phospho-cdc2, 9111S, Cell Signaling Technology), p38 (9212S, Cell Signaling Technology), phosphorylated MK-2 (anti-P-MAPKAPK-2 T334 (27B7), 3007, Cell Signaling Technology), MK-2 (anti-MAPKAPK-2 (D1E11), 12155, Cell Signaling Technology), TFEB (4240S, Cell Signaling Technology), phosphorylated TFEB (anti-P-TFEB S211, 37681, Cell Signaling Technology), or actin (A1978, Sigma). ..

    other:

    Article Title: The Ccr4-Not complex regulates TORC1 signaling and mitochondrial metabolism by promoting vacuole V-ATPase activity
    Article Snippet: AntibodiesThe antibodies used are as follows: rabbit α-RPS6 (ab40820) and rabbit α-Vph1 (ab113683) from Abcam; rabbit α-Phospho S6 (#2211), rabbit α-Phospho-p44/42 MAPK (#4370), and rabbit α-Phospho-AMPKa (#9211) from Cell Signaling Technology; mouse α-Mpk1 (sc-133189), mouse α-Hog1 (sc-165978), mouse α-Myc clone 9E10 (sc-40), and mouse α-HA clone F-7 (sc-7392) from Santa Cruz Biotechnology; mouse α-GFP (Y1030) from UBPBio; and rabbit α-G6PDH (A9521) from Sigma-Aldrich.

    SDS Page:

    Article Title: Iron alters the cell wall composition and intracellular lactate to affect Candida albicans susceptibility to antifungals and host immune response
    Article Snippet: .. Total protein concentrations were determined using the RC DCTM protein assay kit (Bio-Rad) and normalized protein content (30 µg) was loaded in each well for SDS-PAGE. α-Phospho-p42/44 MAPK ERK1/2 Thr-202/Tyr-204 rabbit mAb (Cell Signaling Technology) was used for Cek1-P detection and Hog1 antibody (D3: sc-165978 from Santa Cruz Biotechnology, Inc.) was used for detection of Hog1 protein, as the primary antibody. ..

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  • 96
    Santa Cruz Biotechnology anti hog1 antibody
    Ptc2 inactivates <t>Hog1</t> in vitro. (A) Activated GST-Hog1 was isolated from osmotic stressed yeast and was untreated or treated with Ptc2. Approximately 0.5 μg of GST-Hog1 bound to resin was incubated with 0 to 1.2 μg of Ptc2 for 30 min at 30°C. The resin was washed extensively to remove Ptc2, and GST-Hog1 was incubated with MBP and [γ- 32 P]ATP to assess Hog1 kinase activity. Radiolabel incorporated into MBP was examined by PhosphorImager analysis. (B) Ptc2 inactivates Hog1 by dephosphorylating the phosphothreonine residue (pT) in the activation loop. GST-Hog1 was phosphorylated in vitro, and the sample was untreated (left) or incubated with Ptc2 (right) prior to phosphoamino acid analysis. The radiolabeled amino acids were detected with the PhosphorImager. Arrows, positions of phosphoamino acid standards as revealed by ninhydrin staining.
    Anti Hog1 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti hog1 antibody/product/Santa Cruz Biotechnology
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    86
    Santa Cruz Biotechnology rabbit polyclonal hog1 antibody
    C. neoformans ). Each protein sequence was retrieved from the genome database and NCBI [ S. cerevisiae , Rad53; C. albicans , Rad53; C. neoformans , Rad53; and S. pombe , Cds1] aa, amino acids. (B and C) Phosphorylation of Rad53 was monitored by analysis of the reduced electrophoretic migration using western blotting with anti-FLAG antibody. The Rad53-4xFLAG strain was grown to the mid-logarithmic phase and then treated with MMS (0.02%) for 2 h. The cell extract was incubated at 30°C for 1 h with or without λ-phosphatase (PPase) and PPase inhibitor (B). Rad53 was phosphorylated in response to MMS (0.02%), 4-NQO (0.15 µg/ml), and bleomycin (3 µg/ml) (C). (D) Both Tel1 and Mec1 regulate Rad53 phosphorylation in response to DNA damage stress. WT Rad53-4xFLAG, mec1 Δ Rad53-4xFLAG, tel1 Δ Rad53-4xFLAG, and mec1 Δ tel1 Δ Rad53-4xFLAG strains were treated with MMS (0.02%), and then total protein was extracted from each strain for immunoblot analysis. Rad53 phosphorylation levels were monitored using anti-FLAG antibody. The same blot was stripped and then reprobed with <t>polyclonal</t> <t>anti-Hog1</t> antibody for the loading control. (E) Mec1 and Tel1 play redundant roles in DNA damage stress response in C. neoformans . (Strains: WT Rad53-4xFLAG [YSB3806], Rad53-4xFLAG mec1 Δ [KW102], Rad53-4xFLAG tel1 Δ [KW104], Rad53-4xFLAG mec1 Δ tel1 Δ [KW449], mec1 Δ [YSB3611], tel1 Δ [YSB3844], mec1 Δ tel1 Δ [KW480], rad53 Δ [YSB3785], rad53 Δ+ RAD53 [KW1], and tel1 Δ rad53 Δ [KW106]).
    Rabbit Polyclonal Hog1 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Ptc2 inactivates Hog1 in vitro. (A) Activated GST-Hog1 was isolated from osmotic stressed yeast and was untreated or treated with Ptc2. Approximately 0.5 μg of GST-Hog1 bound to resin was incubated with 0 to 1.2 μg of Ptc2 for 30 min at 30°C. The resin was washed extensively to remove Ptc2, and GST-Hog1 was incubated with MBP and [γ- 32 P]ATP to assess Hog1 kinase activity. Radiolabel incorporated into MBP was examined by PhosphorImager analysis. (B) Ptc2 inactivates Hog1 by dephosphorylating the phosphothreonine residue (pT) in the activation loop. GST-Hog1 was phosphorylated in vitro, and the sample was untreated (left) or incubated with Ptc2 (right) prior to phosphoamino acid analysis. The radiolabeled amino acids were detected with the PhosphorImager. Arrows, positions of phosphoamino acid standards as revealed by ninhydrin staining.

    Journal: Eukaryotic Cell

    Article Title: Role of Ptc2 Type 2C Ser/Thr Phosphatase in Yeast High-Osmolarity Glycerol Pathway Inactivation

    doi: 10.1128/EC.1.6.1032-1040.2002

    Figure Lengend Snippet: Ptc2 inactivates Hog1 in vitro. (A) Activated GST-Hog1 was isolated from osmotic stressed yeast and was untreated or treated with Ptc2. Approximately 0.5 μg of GST-Hog1 bound to resin was incubated with 0 to 1.2 μg of Ptc2 for 30 min at 30°C. The resin was washed extensively to remove Ptc2, and GST-Hog1 was incubated with MBP and [γ- 32 P]ATP to assess Hog1 kinase activity. Radiolabel incorporated into MBP was examined by PhosphorImager analysis. (B) Ptc2 inactivates Hog1 by dephosphorylating the phosphothreonine residue (pT) in the activation loop. GST-Hog1 was phosphorylated in vitro, and the sample was untreated (left) or incubated with Ptc2 (right) prior to phosphoamino acid analysis. The radiolabeled amino acids were detected with the PhosphorImager. Arrows, positions of phosphoamino acid standards as revealed by ninhydrin staining.

    Article Snippet: GST-Ptc2 and GST were detected with an anti-GST antibody (Pharmacia), and Hog1 was visualized with an anti-Hog1 antibody (yC-20; Santa Cruz Biotechnology).

    Techniques: In Vitro, Isolation, Incubation, Activity Assay, Activation Assay, Phosphoamino Acid Analysis, Staining

    Ptc2 inactivates Hog1 in vivo. (A) Overexpression of PTC2 inhibits osmotic-stress-induced Hog1 activation. Hog1 kinase activity in PTC2 overexpressor IMY105, carrying pKT-PTC2 and pHOG1-ha2, and in the control strain, carrying empty vector pKT and pHOG1-ha2, was examined. Before (time zero) and after exposure to osmotic stress (0.4 M NaCl) for various times, Hog1-HA was immunoprecipitated and incubated with MBP and [γ- 32 P]ATP. Radiolabel incorporated into MBP was examined with the PhosphorImager. The graph shows the means of three independent experiments ± standard errors of the means (SEM). (B) Hog1 is hyperactivated in a strain lacking PTC2 and PTC3 . Hog1 kinase activity was monitored prior to and following osmotic stress in a hog1 Δ strain (IMY100) and in a ptc2 Δ ptc3 Δ hog1 Δ strain (CAY9), each carrying a Hog1-HA-expressing plasmid. Hog1 kinase activity was monitored as described for panel A. The graph shows the means of six independent experiments ± SEM. (C) Ptc2 does not inactivate Pbs2 in vivo. Hog1-pY in the 334 strain carrying a plasmid overexpressing PTC2 or an empty vector was examined. The level of Hog1-pY prior to and following osmotic stress was monitored by immunoblotting with an anti-pY antibody. Total Hog1 protein was examined by blotting with an anti-Hog1 antibody.

    Journal: Eukaryotic Cell

    Article Title: Role of Ptc2 Type 2C Ser/Thr Phosphatase in Yeast High-Osmolarity Glycerol Pathway Inactivation

    doi: 10.1128/EC.1.6.1032-1040.2002

    Figure Lengend Snippet: Ptc2 inactivates Hog1 in vivo. (A) Overexpression of PTC2 inhibits osmotic-stress-induced Hog1 activation. Hog1 kinase activity in PTC2 overexpressor IMY105, carrying pKT-PTC2 and pHOG1-ha2, and in the control strain, carrying empty vector pKT and pHOG1-ha2, was examined. Before (time zero) and after exposure to osmotic stress (0.4 M NaCl) for various times, Hog1-HA was immunoprecipitated and incubated with MBP and [γ- 32 P]ATP. Radiolabel incorporated into MBP was examined with the PhosphorImager. The graph shows the means of three independent experiments ± standard errors of the means (SEM). (B) Hog1 is hyperactivated in a strain lacking PTC2 and PTC3 . Hog1 kinase activity was monitored prior to and following osmotic stress in a hog1 Δ strain (IMY100) and in a ptc2 Δ ptc3 Δ hog1 Δ strain (CAY9), each carrying a Hog1-HA-expressing plasmid. Hog1 kinase activity was monitored as described for panel A. The graph shows the means of six independent experiments ± SEM. (C) Ptc2 does not inactivate Pbs2 in vivo. Hog1-pY in the 334 strain carrying a plasmid overexpressing PTC2 or an empty vector was examined. The level of Hog1-pY prior to and following osmotic stress was monitored by immunoblotting with an anti-pY antibody. Total Hog1 protein was examined by blotting with an anti-Hog1 antibody.

    Article Snippet: GST-Ptc2 and GST were detected with an anti-GST antibody (Pharmacia), and Hog1 was visualized with an anti-Hog1 antibody (yC-20; Santa Cruz Biotechnology).

    Techniques: In Vivo, Over Expression, Activation Assay, Activity Assay, Plasmid Preparation, Immunoprecipitation, Incubation, Expressing

    The temperature sensitivity of strains lacking PTC s and PTP2 is due to HOG1. (A) Strains lacking PTC s and PTP2 exhibit growth defects at 37°C. The growth of ptp2 Δ (IMY21a), ptc2 Δ ptc3 Δ (IMY124), ptc3 Δ ptp2 Δ (IMY127b), ptc2 Δ ptc3 Δ ptp2 Δ (IMY128), and wild-type (BBY45) strains on rich medium (yeast extract-peptone-dextrose [YPD]) at 37 and 30°C was compared. (B) The growth defects of the ptc3 Δ ptp2 Δ and ptc2 Δ ptc3 Δ ptp2 Δ strains at 37°C were suppressed by deleting HOG1 . The strains lacking HOG1 were JMY1 ( ptc3 Δ ptp2 Δ hog1 Δ and JMY2 ( ptc2 Δ ptc3 Δ ptp2 Δ hog1 Δ) and were compared on YPD at 37 and 30°C.

    Journal: Eukaryotic Cell

    Article Title: Role of Ptc2 Type 2C Ser/Thr Phosphatase in Yeast High-Osmolarity Glycerol Pathway Inactivation

    doi: 10.1128/EC.1.6.1032-1040.2002

    Figure Lengend Snippet: The temperature sensitivity of strains lacking PTC s and PTP2 is due to HOG1. (A) Strains lacking PTC s and PTP2 exhibit growth defects at 37°C. The growth of ptp2 Δ (IMY21a), ptc2 Δ ptc3 Δ (IMY124), ptc3 Δ ptp2 Δ (IMY127b), ptc2 Δ ptc3 Δ ptp2 Δ (IMY128), and wild-type (BBY45) strains on rich medium (yeast extract-peptone-dextrose [YPD]) at 37 and 30°C was compared. (B) The growth defects of the ptc3 Δ ptp2 Δ and ptc2 Δ ptc3 Δ ptp2 Δ strains at 37°C were suppressed by deleting HOG1 . The strains lacking HOG1 were JMY1 ( ptc3 Δ ptp2 Δ hog1 Δ and JMY2 ( ptc2 Δ ptc3 Δ ptp2 Δ hog1 Δ) and were compared on YPD at 37 and 30°C.

    Article Snippet: GST-Ptc2 and GST were detected with an anti-GST antibody (Pharmacia), and Hog1 was visualized with an anti-Hog1 antibody (yC-20; Santa Cruz Biotechnology).

    Techniques:

    (A) Hydrogen peroxide activation of RLM1 transcription in mpk1Δ strain. Wild-type strain (Dl100) (▪) and the isogenic mpk1 Δ (▴) strain were transformed with px2RLM1 and then plated in 24-well plates on media containing the β-galactosidase substrate CPRG as described in Materials and Methods . Hydrogen peroxide was added to the media in increasing concentrations (0.1–0.5 mM), and β-galactosidase activity was measured at 570 nm after 24 h. Results shown are from five independent assays. (B) RLM1 transcription induction in hog1Δ strain. Wild-type strain (EY957) (▪) and the isogenic hog1 Δ strain (•) were transformed with px2RLM1 and then plated in 24-well plates on media containing the β-galactosidase substrate CPRG as described in Materials and Methods . Hydrogen peroxide was added to the media in increasing concentrations (0.1–0.5 mM), and β-galactosidase activity was measured at 570 nm after 24 h. Results shown are from five independent assays.

    Journal: Molecular Biology of the Cell

    Article Title: Oxidative Stress Activates FUS1 and RLM1 Transcription in the Yeast Saccharomyces cerevisiae in an Oxidant-dependent Manner D⃞

    doi: 10.1091/mbc.E04-02-0142

    Figure Lengend Snippet: (A) Hydrogen peroxide activation of RLM1 transcription in mpk1Δ strain. Wild-type strain (Dl100) (▪) and the isogenic mpk1 Δ (▴) strain were transformed with px2RLM1 and then plated in 24-well plates on media containing the β-galactosidase substrate CPRG as described in Materials and Methods . Hydrogen peroxide was added to the media in increasing concentrations (0.1–0.5 mM), and β-galactosidase activity was measured at 570 nm after 24 h. Results shown are from five independent assays. (B) RLM1 transcription induction in hog1Δ strain. Wild-type strain (EY957) (▪) and the isogenic hog1 Δ strain (•) were transformed with px2RLM1 and then plated in 24-well plates on media containing the β-galactosidase substrate CPRG as described in Materials and Methods . Hydrogen peroxide was added to the media in increasing concentrations (0.1–0.5 mM), and β-galactosidase activity was measured at 570 nm after 24 h. Results shown are from five independent assays.

    Article Snippet: Anti-Mpk1 and anti-Hog1 antibodies both from Santa Cruz Biotechnology (Santa Cruz, CA) were used at a final dilution 1:1000.

    Techniques: Activation Assay, Transformation Assay, Activity Assay

    (A) Mpk1 is phosphorylated on exposure of cells to hydrogen peroxide. Mid-log cultures of wild-type strain cells (EY957) were treated with hydrogen peroxide at 5 mM final concentration with 50 nM α-pheromone as a positive control. Samples were taken at the indicated times (0, 15, and 30 min). Wild-type cells were treated with α-pheromone for 60 min. Hydrogen peroxide was added to mpk1 Δ strain for 15 min. Western blot analysis was performed as described in Materials and Methods by using anti-phospho-p44/p42 antibody to detect the phosphorylated Mpk1, Kss1 and Fus3, anti-Mpk1 to detect the total protein and anti-Hog1 as a loading control. (B) Hydrogen peroxide activates Hog1 MAPK. Mid-log cultures of wild-type strain cells (EY957) were treated with hydrogen peroxide at 5 mM final concentration. Samples were taken at the indicated times (0, 15, and 30 min). Strain disrupted in HOG1 gene, used as negative control, was treated with hydrogen peroxide for 15 min. Western blot analysis was performed as described in Materials and Methods by using anti-phospho-p38 antibody to detect the phosphorylated Hog1, with anti-Hog1 to detect the total protein and anti-Mpk1 as a loading control.

    Journal: Molecular Biology of the Cell

    Article Title: Oxidative Stress Activates FUS1 and RLM1 Transcription in the Yeast Saccharomyces cerevisiae in an Oxidant-dependent Manner D⃞

    doi: 10.1091/mbc.E04-02-0142

    Figure Lengend Snippet: (A) Mpk1 is phosphorylated on exposure of cells to hydrogen peroxide. Mid-log cultures of wild-type strain cells (EY957) were treated with hydrogen peroxide at 5 mM final concentration with 50 nM α-pheromone as a positive control. Samples were taken at the indicated times (0, 15, and 30 min). Wild-type cells were treated with α-pheromone for 60 min. Hydrogen peroxide was added to mpk1 Δ strain for 15 min. Western blot analysis was performed as described in Materials and Methods by using anti-phospho-p44/p42 antibody to detect the phosphorylated Mpk1, Kss1 and Fus3, anti-Mpk1 to detect the total protein and anti-Hog1 as a loading control. (B) Hydrogen peroxide activates Hog1 MAPK. Mid-log cultures of wild-type strain cells (EY957) were treated with hydrogen peroxide at 5 mM final concentration. Samples were taken at the indicated times (0, 15, and 30 min). Strain disrupted in HOG1 gene, used as negative control, was treated with hydrogen peroxide for 15 min. Western blot analysis was performed as described in Materials and Methods by using anti-phospho-p38 antibody to detect the phosphorylated Hog1, with anti-Hog1 to detect the total protein and anti-Mpk1 as a loading control.

    Article Snippet: Anti-Mpk1 and anti-Hog1 antibodies both from Santa Cruz Biotechnology (Santa Cruz, CA) were used at a final dilution 1:1000.

    Techniques: Concentration Assay, Positive Control, Western Blot, Negative Control

    Model of the yeast hyperosmotic-response MAPK pathway. (A) The osmotic signals from Sho1 or Sln1 are transduced by unique components and converge to activate Pbs2. The Sho1 branch requires Cdc42, Ste20, and Ste50 to activate Ste11. The Sln1 protein activates Ssk2 and Ssk22 through Ypd1 and Ssk1. Any of Ste11, Ssk2, or Ssk22 is able to activate Pbs2, which then phosphorylates Hog1, resulting in translocation of Hog1 to the nucleus that regulates responsible genes expression for osmoadaptation. (B) The schematic diagram of Sho1 and Sln1 orthologs in Botrytis cinerea . The functional domains were retrieved in EnsemblFungi ( https://fungi.ensembl.org/Botrytis_cinerea/Info/Index ). (C) Subcellular localization of BcSho1-GFP and BcSln1-GFP fusion proteins in B. cienrea . Bars, 10 μm.

    Journal: Frontiers in Microbiology

    Article Title: The Sensor Proteins BcSho1 and BcSln1 Are Involved in, Though Not Essential to, Vegetative Differentiation, Pathogenicity and Osmotic Stress Tolerance in Botrytis cinerea

    doi: 10.3389/fmicb.2019.00328

    Figure Lengend Snippet: Model of the yeast hyperosmotic-response MAPK pathway. (A) The osmotic signals from Sho1 or Sln1 are transduced by unique components and converge to activate Pbs2. The Sho1 branch requires Cdc42, Ste20, and Ste50 to activate Ste11. The Sln1 protein activates Ssk2 and Ssk22 through Ypd1 and Ssk1. Any of Ste11, Ssk2, or Ssk22 is able to activate Pbs2, which then phosphorylates Hog1, resulting in translocation of Hog1 to the nucleus that regulates responsible genes expression for osmoadaptation. (B) The schematic diagram of Sho1 and Sln1 orthologs in Botrytis cinerea . The functional domains were retrieved in EnsemblFungi ( https://fungi.ensembl.org/Botrytis_cinerea/Info/Index ). (C) Subcellular localization of BcSho1-GFP and BcSln1-GFP fusion proteins in B. cienrea . Bars, 10 μm.

    Article Snippet: In addition, the samples were detected with anti-Hog1 antibody (C-terminal anti-Hog1) (Santa Cruz Biotechnology, Santa Cruz, CA, United States) as a reference.

    Techniques: Translocation Assay, Expressing, Functional Assay

    C. neoformans ). Each protein sequence was retrieved from the genome database and NCBI [ S. cerevisiae , Rad53; C. albicans , Rad53; C. neoformans , Rad53; and S. pombe , Cds1] aa, amino acids. (B and C) Phosphorylation of Rad53 was monitored by analysis of the reduced electrophoretic migration using western blotting with anti-FLAG antibody. The Rad53-4xFLAG strain was grown to the mid-logarithmic phase and then treated with MMS (0.02%) for 2 h. The cell extract was incubated at 30°C for 1 h with or without λ-phosphatase (PPase) and PPase inhibitor (B). Rad53 was phosphorylated in response to MMS (0.02%), 4-NQO (0.15 µg/ml), and bleomycin (3 µg/ml) (C). (D) Both Tel1 and Mec1 regulate Rad53 phosphorylation in response to DNA damage stress. WT Rad53-4xFLAG, mec1 Δ Rad53-4xFLAG, tel1 Δ Rad53-4xFLAG, and mec1 Δ tel1 Δ Rad53-4xFLAG strains were treated with MMS (0.02%), and then total protein was extracted from each strain for immunoblot analysis. Rad53 phosphorylation levels were monitored using anti-FLAG antibody. The same blot was stripped and then reprobed with polyclonal anti-Hog1 antibody for the loading control. (E) Mec1 and Tel1 play redundant roles in DNA damage stress response in C. neoformans . (Strains: WT Rad53-4xFLAG [YSB3806], Rad53-4xFLAG mec1 Δ [KW102], Rad53-4xFLAG tel1 Δ [KW104], Rad53-4xFLAG mec1 Δ tel1 Δ [KW449], mec1 Δ [YSB3611], tel1 Δ [YSB3844], mec1 Δ tel1 Δ [KW480], rad53 Δ [YSB3785], rad53 Δ+ RAD53 [KW1], and tel1 Δ rad53 Δ [KW106]).

    Journal: mBio

    Article Title: Rad53- and Chk1-Dependent DNA Damage Response Pathways Cooperatively Promote Fungal Pathogenesis and Modulate Antifungal Drug Susceptibility

    doi: 10.1128/mBio.01726-18

    Figure Lengend Snippet: C. neoformans ). Each protein sequence was retrieved from the genome database and NCBI [ S. cerevisiae , Rad53; C. albicans , Rad53; C. neoformans , Rad53; and S. pombe , Cds1] aa, amino acids. (B and C) Phosphorylation of Rad53 was monitored by analysis of the reduced electrophoretic migration using western blotting with anti-FLAG antibody. The Rad53-4xFLAG strain was grown to the mid-logarithmic phase and then treated with MMS (0.02%) for 2 h. The cell extract was incubated at 30°C for 1 h with or without λ-phosphatase (PPase) and PPase inhibitor (B). Rad53 was phosphorylated in response to MMS (0.02%), 4-NQO (0.15 µg/ml), and bleomycin (3 µg/ml) (C). (D) Both Tel1 and Mec1 regulate Rad53 phosphorylation in response to DNA damage stress. WT Rad53-4xFLAG, mec1 Δ Rad53-4xFLAG, tel1 Δ Rad53-4xFLAG, and mec1 Δ tel1 Δ Rad53-4xFLAG strains were treated with MMS (0.02%), and then total protein was extracted from each strain for immunoblot analysis. Rad53 phosphorylation levels were monitored using anti-FLAG antibody. The same blot was stripped and then reprobed with polyclonal anti-Hog1 antibody for the loading control. (E) Mec1 and Tel1 play redundant roles in DNA damage stress response in C. neoformans . (Strains: WT Rad53-4xFLAG [YSB3806], Rad53-4xFLAG mec1 Δ [KW102], Rad53-4xFLAG tel1 Δ [KW104], Rad53-4xFLAG mec1 Δ tel1 Δ [KW449], mec1 Δ [YSB3611], tel1 Δ [YSB3844], mec1 Δ tel1 Δ [KW480], rad53 Δ [YSB3785], rad53 Δ+ RAD53 [KW1], and tel1 Δ rad53 Δ [KW106]).

    Article Snippet: To monitor Hog1 protein levels as the loading control, a primary rabbit polyclonal Hog1 antibody (SC-9079; Santa Cruz Biotechnology) and a secondary anti-rabbit IgG horseradish peroxidase-conjugated antibody (A6154; Sigma) were used.

    Techniques: Sequencing, Migration, Western Blot, Incubation