Journal: CNS Neuroscience & Therapeutics

Article Title: Downregulation of microRNA‐124‐3p promotes subventricular zone neural stem cell activation by enhancing the function of BDNF downstream pathways after traumatic brain injury in adult rats

doi: 10.1111/cns.13845

Figure Lengend Snippet: Primary antibodies used in IF and WB

Article Snippet: Phospho‐p44/42 MAK , CST , #4370 , AB_2315112 , 1:1000 , WB.

Techniques:

Journal: Frontiers in Microbiology

Article Title: Coordinated Regulation of Protoperithecium Development by MAP Kinases MAK-1 and MAK-2 in Neurospora crassa

doi: 10.3389/fmicb.2021.769615

Figure Lengend Snippet: Genes responsive to sexual development induction are regulated by MAK-1 and MAK-2 proteins. (A) A schematic representation of sample preparation used for RNA sequencing and RT-qPCR. (B) Number of genes whose expressions were up- and downregulated during protoperithecium development (PP3.5) in wild type. (C) Venn analysis of genes downregulated in Δ mak-1 and Δ mak-2 mutants. Genes selected are among 2,894 genes that were upregulated in protoperithecia compared with hyphae in wild type, but their upregulation was attenuated in Δ mak-1 and Δ mak-2 mutants. (D) Venn analysis of genes upregulated in Δ mak-1 and Δ mak-2 mutants. Genes selected are among 655 genes that were downregulated in protoperithecia compared with hyphae in wild type, but their downregulation was weakened in Δ mak-1 and Δ mak-2 mutants.

Article Snippet: To analyze the phosphorylation status of the MAK-1 and MAK-2 proteins, a polyclonal rabbit phospho-p44/42 MAP kinase antiserum (Cell Signaling Technology, United States) was used as a primary antibody, and AffiniPure goat anti-rabbit immunoglobulin G (IgG) (H+L) (APPLYGEN) was used as the secondary antibody ( ; ).

Techniques: Sample Prep, RNA Sequencing Assay, Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: Coordinated Regulation of Protoperithecium Development by MAP Kinases MAK-1 and MAK-2 in Neurospora crassa

doi: 10.3389/fmicb.2021.769615

Figure Lengend Snippet: Confirmation of impacts of mak-1 and mak-2 in transcriptional responses by selected genes to sexual development induction through RT-qPCR analysis. Hyphae: samples of N. crassa in vegetative growth stage; PP3.5: samples of N. crassa after 3.5 days of sexual development induction; PP5.5: samples of N. crassa after 5.5 days of sexual development induction. Relative gene expression in Δ mak-1 and Δ mak-2 mutants vs. wild type (WT) was calculated using 2 – ΔΔ CT method. All expression levels were normalized to expression level of β-tubulin. Bars represent mean and standard error for three replicates. Analysis of variance statistically analyzed differences between mutants and wild type. Values with P < 0.001, 0.001 < P < 0.01, and 0.01 < P < 0.05 are marked with *** , ** , and *, respectively.

Article Snippet: To analyze the phosphorylation status of the MAK-1 and MAK-2 proteins, a polyclonal rabbit phospho-p44/42 MAP kinase antiserum (Cell Signaling Technology, United States) was used as a primary antibody, and AffiniPure goat anti-rabbit immunoglobulin G (IgG) (H+L) (APPLYGEN) was used as the secondary antibody ( ; ).

Techniques: Quantitative RT-PCR, Expressing

Journal: Frontiers in Microbiology

Article Title: Coordinated Regulation of Protoperithecium Development by MAP Kinases MAK-1 and MAK-2 in Neurospora crassa

doi: 10.3389/fmicb.2021.769615

Figure Lengend Snippet: Melanin biosynthetic genes are involved in sexual development of N . crassa . (A) RT-qPCR analysis of mld - 1 , tnr-2 , tnr-1 , and scy-1 genes. Hyphae: samples of N. crassa in vegetative growth stage; PP3.5: samples of N. crassa after 3.5 days of sexual development induction; PP5.5: samples of N. crassa after 5.5 days of sexual development induction. Relative gene expression in Δ mld - 1 , Δ mak-1 , and Δ mak-2 mutants vs. wild-type strain was calculated using 2 – ΔΔ CT method. All expression levels were normalized to expression of β-tubulin. Bars represent mean and standard error for three replicates. Analysis of variance statistically analyzed differences between mutants and wild type. Values with P < 0.001, 0.001 < P < 0.01, and 0.01 < P < 0.05 are marked with *** , ** , and *, respectively. (B) Perithecium and ascospores production in Δ mld - 1 , Δ tnr-2 , Δ tnr-1 , and Δ scy mutants. Δ tnr-1 and Δ scy mutants cannot form mature asci. Mutants and wild type were used as female parent and first grown on solid crossing medium for 5.5 days under constant darkness at 25°C. Then, a wild-type strain with opposite mating type, as male parent, was inoculated on colony surface of female strains and incubated at 25°C for another 7 days under constant darkness. Perithecia and ascospores formation were checked and imaged.

Article Snippet: To analyze the phosphorylation status of the MAK-1 and MAK-2 proteins, a polyclonal rabbit phospho-p44/42 MAP kinase antiserum (Cell Signaling Technology, United States) was used as a primary antibody, and AffiniPure goat anti-rabbit immunoglobulin G (IgG) (H+L) (APPLYGEN) was used as the secondary antibody ( ; ).

Techniques: Quantitative RT-PCR, Expressing, Incubation

Journal: Frontiers in Microbiology

Article Title: Coordinated Regulation of Protoperithecium Development by MAP Kinases MAK-1 and MAK-2 in Neurospora crassa

doi: 10.3389/fmicb.2021.769615

Figure Lengend Snippet: Total protein and phosphorylation status of MAK-1 and MAK-2 in deletion mutants of kinases belonging to PR- and CWI-MAP kinase pathways during protoperithecium morphogenesis. Total protein levels of MAK-1 and MAK-2 were detected by Western blot analysis with antibody Kss1 and Fus3 (Santa Cruz), respectively. Phosphorylation status of MAK-1 and MAK-2 were detected by phospho-p44/42 MAPK antibody (Cell signaling). β-Tubulin was also detected as loading control. Hyphae: samples of N. crassa in vegetative growth stage; PP3.5: samples of N. crassa after 3.5 days of sexual development induction; PP5.5: samples of N. crassa after 5.5 days of sexual development induction.

Article Snippet: To analyze the phosphorylation status of the MAK-1 and MAK-2 proteins, a polyclonal rabbit phospho-p44/42 MAP kinase antiserum (Cell Signaling Technology, United States) was used as a primary antibody, and AffiniPure goat anti-rabbit immunoglobulin G (IgG) (H+L) (APPLYGEN) was used as the secondary antibody ( ; ).

Techniques: Western Blot

Journal: Frontiers in Microbiology

Article Title: Coordinated Regulation of Protoperithecium Development by MAP Kinases MAK-1 and MAK-2 in Neurospora crassa

doi: 10.3389/fmicb.2021.769615

Figure Lengend Snippet: Genes responsive to sexual development induction are regulated by MAK-1 and MAK-2 proteins. (A) A schematic representation of sample preparation used for RNA sequencing and RT-qPCR. (B) Number of genes whose expressions were up- and downregulated during protoperithecium development (PP3.5) in wild type. (C) Venn analysis of genes downregulated in Δ mak-1 and Δ mak-2 mutants. Genes selected are among 2,894 genes that were upregulated in protoperithecia compared with hyphae in wild type, but their upregulation was attenuated in Δ mak-1 and Δ mak-2 mutants. (D) Venn analysis of genes upregulated in Δ mak-1 and Δ mak-2 mutants. Genes selected are among 655 genes that were downregulated in protoperithecia compared with hyphae in wild type, but their downregulation was weakened in Δ mak-1 and Δ mak-2 mutants.

Article Snippet: To analyze the phosphorylation status of the MAK-1 and MAK-2 proteins, a polyclonal rabbit phospho-p44/42 MAP kinase antiserum (Cell Signaling Technology, United States) was used as a primary antibody, and AffiniPure goat anti-rabbit immunoglobulin G (IgG) (H+L) (APPLYGEN) was used as the secondary antibody ( ; ).

Techniques: Sample Prep, RNA Sequencing Assay, Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: Coordinated Regulation of Protoperithecium Development by MAP Kinases MAK-1 and MAK-2 in Neurospora crassa

doi: 10.3389/fmicb.2021.769615

Figure Lengend Snippet: Confirmation of impacts of mak-1 and mak-2 in transcriptional responses by selected genes to sexual development induction through RT-qPCR analysis. Hyphae: samples of N. crassa in vegetative growth stage; PP3.5: samples of N. crassa after 3.5 days of sexual development induction; PP5.5: samples of N. crassa after 5.5 days of sexual development induction. Relative gene expression in Δ mak-1 and Δ mak-2 mutants vs. wild type (WT) was calculated using 2 – ΔΔ CT method. All expression levels were normalized to expression level of β-tubulin. Bars represent mean and standard error for three replicates. Analysis of variance statistically analyzed differences between mutants and wild type. Values with P < 0.001, 0.001 < P < 0.01, and 0.01 < P < 0.05 are marked with *** , ** , and *, respectively.

Article Snippet: To analyze the phosphorylation status of the MAK-1 and MAK-2 proteins, a polyclonal rabbit phospho-p44/42 MAP kinase antiserum (Cell Signaling Technology, United States) was used as a primary antibody, and AffiniPure goat anti-rabbit immunoglobulin G (IgG) (H+L) (APPLYGEN) was used as the secondary antibody ( ; ).

Techniques: Quantitative RT-PCR, Expressing

Journal: Frontiers in Microbiology

Article Title: Coordinated Regulation of Protoperithecium Development by MAP Kinases MAK-1 and MAK-2 in Neurospora crassa

doi: 10.3389/fmicb.2021.769615

Figure Lengend Snippet: Melanin biosynthetic genes are involved in sexual development of N . crassa . (A) RT-qPCR analysis of mld - 1 , tnr-2 , tnr-1 , and scy-1 genes. Hyphae: samples of N. crassa in vegetative growth stage; PP3.5: samples of N. crassa after 3.5 days of sexual development induction; PP5.5: samples of N. crassa after 5.5 days of sexual development induction. Relative gene expression in Δ mld - 1 , Δ mak-1 , and Δ mak-2 mutants vs. wild-type strain was calculated using 2 – ΔΔ CT method. All expression levels were normalized to expression of β-tubulin. Bars represent mean and standard error for three replicates. Analysis of variance statistically analyzed differences between mutants and wild type. Values with P < 0.001, 0.001 < P < 0.01, and 0.01 < P < 0.05 are marked with *** , ** , and *, respectively. (B) Perithecium and ascospores production in Δ mld - 1 , Δ tnr-2 , Δ tnr-1 , and Δ scy mutants. Δ tnr-1 and Δ scy mutants cannot form mature asci. Mutants and wild type were used as female parent and first grown on solid crossing medium for 5.5 days under constant darkness at 25°C. Then, a wild-type strain with opposite mating type, as male parent, was inoculated on colony surface of female strains and incubated at 25°C for another 7 days under constant darkness. Perithecia and ascospores formation were checked and imaged.

Article Snippet: To analyze the phosphorylation status of the MAK-1 and MAK-2 proteins, a polyclonal rabbit phospho-p44/42 MAP kinase antiserum (Cell Signaling Technology, United States) was used as a primary antibody, and AffiniPure goat anti-rabbit immunoglobulin G (IgG) (H+L) (APPLYGEN) was used as the secondary antibody ( ; ).

Techniques: Quantitative RT-PCR, Expressing, Incubation

Journal: Frontiers in Microbiology

Article Title: Coordinated Regulation of Protoperithecium Development by MAP Kinases MAK-1 and MAK-2 in Neurospora crassa

doi: 10.3389/fmicb.2021.769615

Figure Lengend Snippet: Total protein and phosphorylation status of MAK-1 and MAK-2 in deletion mutants of kinases belonging to PR- and CWI-MAP kinase pathways during protoperithecium morphogenesis. Total protein levels of MAK-1 and MAK-2 were detected by Western blot analysis with antibody Kss1 and Fus3 (Santa Cruz), respectively. Phosphorylation status of MAK-1 and MAK-2 were detected by phospho-p44/42 MAPK antibody (Cell signaling). β-Tubulin was also detected as loading control. Hyphae: samples of N. crassa in vegetative growth stage; PP3.5: samples of N. crassa after 3.5 days of sexual development induction; PP5.5: samples of N. crassa after 5.5 days of sexual development induction.

Article Snippet: To analyze the phosphorylation status of the MAK-1 and MAK-2 proteins, a polyclonal rabbit phospho-p44/42 MAP kinase antiserum (Cell Signaling Technology, United States) was used as a primary antibody, and AffiniPure goat anti-rabbit immunoglobulin G (IgG) (H+L) (APPLYGEN) was used as the secondary antibody ( ; ).

Techniques: Western Blot