Journal: Plant Physiology

Article Title: Decreased Protein Abundance of Lycopene β -Cyclase Contributes to Red Flesh in Domesticated Watermelon ¹

doi: 10.1104/pp.19.01409

Figure Lengend Snippet: Catalytic function and localization of three ClLCYB proteins. A, Three ClLCYB haplotypes found in watermelons with different flesh colors: ClLCYBred with Val (V) at residue 226 and Lys (K) at residue 435 in all the red-fleshed lines; ClLCYByellow with Phe (F) at residue 226 and Asn (N) at residue 435 in the cultivated lines that did not accumulate lycopene, such as cv CS and cv JLM; and ClLCYBwhite with Phe (F) at residue 226 and Lys (K) at residue 435 in wild and semiwild white-fleshed watermelons. B, Subcellular localization of three ClLCYBs. ClLCYB-mCherry (ClLCYBwhite, ClLCYByellow, and ClLCYBred) and mCherry vector (vector control) were transiently produced in watermelon fruit protoplasts incubated in the dark for 12 h. The protoplasts were observed under chromoplast autofluorescence (column 1), under bright field (column 2), under mCherry fluorescence (column 3), and in merged images (column 4) with a confocal microscope. Bars = 20 µm. C, HPLC analysis of the color complementation of a pAC-LYC bacterial line with three ClLCYB constructs. The pAC-LYC plasmid (Cunningham and Gantt, 2007) enables lycopene production and accumulation in E. coli. pET28a expression vectors (Promega) carrying the three ClLCYB constructs were transformed into pAC-LYC cells, with empty pET28a as a control. Carotenoid pigment composition was examined in cultures of E. coli containing the plasmid indicated above. HPLC detection was performed at 440 nm. D, Michaelis-Menten profiles for the activities of three ClLCYBs and the concentration of the substrate lycopene in 5 mm NADPH, pH 6.5, at 25°C.

Article Snippet: The pAC-LYC plasmid ( Cunningham and Gantt, 2007 ) enables lycopene production and accumulation in E. coli . pET28a expression vectors (Promega) carrying the three ClLCYB constructs were transformed into pAC-LYC cells, with empty pET28a as a control.

Techniques: Residue, Plasmid Preparation, Control, Produced, Incubation, Fluorescence, Microscopy, Construct, Expressing, Transformation Assay, Concentration Assay

Journal: Frontiers in Microbiology

Article Title: The Protocatechuate 3,4-Dioxygenase Solubility (PCDS) Tag Enhances the Expression and Solubility of Heterogenous Proteins in Escherichia coli

doi: 10.3389/fmicb.2021.779541

Figure Lengend Snippet: Determination of the expression of recombinant PcaHG in E . coli BL21 (DE3) by SDS-PAGE. Total cell extract and soluble cell extract from induced E . coli BL21 (DE3). Lane M, protein molecular mass standards (Unstained Protein Molecular Weight Marker, No. 26610, Thermo Fisher Scientific, Waltham, MA, United States). (A) lane 1, total BL21 (DE3)/pET-28a (+), lane 2, soluble BL21 (DE3)/pET-28a (+), lane 3, total BL21 (DE3)/pET-28a- pcaHG98e01 , lane 4, soluble BL21 (DE3)/pET-28a- pcaHG98e01 , lane 5, total BL21 (DE3)/pET-28a- pcaHG98e02 , lane 6, soluble BL21 (DE3)/pET-28a- pcaHG98e02 . (B) lane 1, total BL21 (DE3)/pET-28a (+), lane 2, soluble BL21 (DE3)/pET-28a (+), lane 3, total BL21 (DE3)/pET-28a - pcaHG98e03 , lane 4, soluble BL21 (DE3)/pET-28a- pcaHG98e03 , lane 5, total BL21 (DE3)/pET-28a- pcaHG98e04 , lane 6, soluble BL21 (DE3)/pET-28a- pcaHG98e04 . (C) lane 1, soluble BL21 (DE3)/pET-28a- pcaHG98e04 ; lane 2. Purification of His 6 -PcaHG98e04-PCDS protein from BL21 (DE3)/pET-28a- pcaHG98e04 .

Article Snippet: pET-28a- pcaHG98e02 , Expression vector for pcaHG , PCR amplified with primers pcahg9801 and pcahg9802 from strain NCIMB 9866 genome, inserted into the Nde I- Hin d III restriction sites of pET-28a (+) , This study.

Techniques: Expressing, Recombinant, SDS Page, Molecular Weight, Marker, Purification

Journal: Frontiers in Microbiology

Article Title: The Protocatechuate 3,4-Dioxygenase Solubility (PCDS) Tag Enhances the Expression and Solubility of Heterogenous Proteins in Escherichia coli

doi: 10.3389/fmicb.2021.779541

Figure Lengend Snippet: Determination of pcaHG98 -encoded PCA 3,4 dioxygenase activity. Spectrophotometric changes during oxidation of PCA by pcaHG98 -encoded PCA 3,4 dioxygenase. The assay of PCA catalyzed by cell extracts from E . coli BL21 (DE3) carrying pET-28a- pcaHG98e01 (A) or pET-28a- pcaHG98e04 (D) . There was no enzyme activity in the cell extracts from BL21 (DE3) carrying pET-28a- pcaHG98e02 (B) or pET-28a- pcaHG98e03 (C) .

Article Snippet: pET-28a- pcaHG98e02 , Expression vector for pcaHG , PCR amplified with primers pcahg9801 and pcahg9802 from strain NCIMB 9866 genome, inserted into the Nde I- Hin d III restriction sites of pET-28a (+) , This study.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Cytoplasmic sequestration of the RhoA effector mDiaphanous1 by Prohibitin2 promotes muscle differentiation

doi: 10.1101/283044

Figure Lengend Snippet: (A) Domain structure of full-length (FL) mDia1 and constitutively active mDia1 mutant, mDia1ΔN3. Grey lines indicate RhoA-GTPase and DAD binding regions. G-GTPase binding domain, DID-Diaphanous inhibitory domain, FH1, FH2, FH3-Formin Homology domains, DAD-Diaphanous Auto-inhibitory Domain. Start positions of the domains are depicted. (B) Phb2 identified as mDia1 interacting protein in a yeast two hybrid screen. PJ69-4A was co-transformed with Phb2-AD and mDia1ΔN3-BD (positive GAL4 reconstitution) or empty-BD (negative GAL4 reconstitution) and four colonies per reconstitution were screened for ADE2 and LacZ reporters on -Trp/-Leu/-Ade and -Trp/-Leu+X-Gal plates respectively. Positive control “P”- Drosophila Batman-AD and GAGA factor-BD, negative control “N”-empty-AD and empty BD. Induction of ADE2 is indicated by growth and induction of LacZ is indicated by blue colour. Trp-Tryptophan, Leu-Leucine, Ade-Adenine. AD-Activation domain, BD binding domain. (C) Domain structure of Phb2-FL and Phb2-Y2H. HYD-Hydrophobic region, PHB-Prohibitin domain, CC-Coiled coil domain. (D) Co-IP of exogenous Phb2 and mDia1ΔN3 to confirm the interaction. HEK293T, co-transfected with mDia1ΔN3 and Phb2-Y2H, and pulled down with anti-Flag antibody. (E,F) Reciprocal IP of endogenous mDia1 and Phb2 to identify stage-specific interaction. Lysates from proliferating MB (GM), MT in differentiation medium for 24 (D24), 36, (D36), 72 (D72) and 120 (D120) hours were harvested and subjected to IP with anti-mDia1 (E) or ant-Phb2 (F) antibodies. (G) LC-MS/MS analysis of mDia1-interacting proteins in myoblasts (MB) and myotubes (MT). Venn diagram represents the number of proteins that bind mDia1 in MB or MT or both MB and MT. (H) Phb2 peptides identified in MT lysates by LC-MS/MS analysis of mDia1 IP proteins. Peptides identified in the first biological replicate are indicated in red, peptides identified in the second and third replicate are shown in blue and those peptides common in all three replicates are underlined in Phb2 full-length aa sequence (NCBI Reference Sequence # NP_031557.2). The numbers represent aa position on mDia1 or Phb2 (A, C).

Article Snippet: Expression plasmids for GFP-tagged mouse mDia1, mDia1FL, mDia1ΔN3, mDia1F2, mDia1ΔN3(HindIII), mDia1H+P and mDia1CC were gifts from S Narumiya ( ). mDia1ΔN3-BD was cloned from mDia1ΔN3-pET28a into pGBKT7 (GAL4 binding domain vector-Clonetech) using Nde1 and BamH1.

Techniques: Mutagenesis, Binding Assay, Two Hybrid Screening, Transformation Assay, Positive Control, Negative Control, Activation Assay, Co-Immunoprecipitation Assay, Transfection, Liquid Chromatography with Mass Spectroscopy, Sequencing

Journal: bioRxiv

Article Title: Cytoplasmic sequestration of the RhoA effector mDiaphanous1 by Prohibitin2 promotes muscle differentiation

doi: 10.1101/283044

Figure Lengend Snippet: Yeast two-hybrid screen to identify novel mDia1-interacting proteins. A GAL4 hybrid reconstitution assay with putative mDia1 interactors was performed to study the induction of reporters ADE2 and LacZ on -Trp/-Leu/-Ade and -Trp/-Leu+X-Gal respectively. PJ69-4A was co transformed with interacting protein plasmid and mDia1ΔN3-BD (positive reconstitution) or empty BD vector (negative reconstitution). Four colonies per per reconstitution assay (positive and negative) were screened on selection plates. PJ69-4A co-transformed with bona fide interacting proteins Drosophila Batman-AD and GAGA factor-BD served as a positive control “P” for reporter expression and PJ69-4A co-transformed with empty pACT2 and pGBKT7 vectors served as a negative control “N”. Induction of ADE2 reporter is indicated by growth and induction of LacZ expression is indicated by blue colour in the colonies on the selection plates. Seven library clones that induced the reporter expression were finally selected from the screen as mDia1-interacting proteins of interest. (A) Profilin1 (Pfn1), known interactor for mDia1 (B) Cadherin11 (Cdh11) (C) Niemann Pick Type C2 (Npc2) (D) Leukocyte receptor cluster (LRC) member 8 (Leng8) (E) Growth factor receptor bound protein 2 (Grb2) (F) Protein-kinase, interferon-inducible double stranded RNA dependent inhibitor, repressor of (p58 repressor) (Prkrir) (G) Cytochrome c1 (Cyc1) were identified as mDia1-interacting proteins. Trp-Tryptophan, Leu-Leucine, Ade-Adenine.

Article Snippet: Expression plasmids for GFP-tagged mouse mDia1, mDia1FL, mDia1ΔN3, mDia1F2, mDia1ΔN3(HindIII), mDia1H+P and mDia1CC were gifts from S Narumiya ( ). mDia1ΔN3-BD was cloned from mDia1ΔN3-pET28a into pGBKT7 (GAL4 binding domain vector-Clonetech) using Nde1 and BamH1.

Techniques: Two Hybrid Screening, Reconstitution Assay, Transformation Assay, Plasmid Preparation, Selection, Positive Control, Expressing, Negative Control, Clone Assay

Journal: bioRxiv

Article Title: Cytoplasmic sequestration of the RhoA effector mDiaphanous1 by Prohibitin2 promotes muscle differentiation

doi: 10.1101/283044

Figure Lengend Snippet: (A) Schematic for mouse mDia1 truncation mutants. (B) Western blot to detect expression level of mDia1 mutants. Lysates from HEK293T transfected with mDia1 mutants were probed using anti-GFP antibody. GAPDH was used as a loading control. (C, C’) Co-IP of mDia1 mutants and Phb2 to map interaction domains. HEK293T cells were transfected with Phb2-Y2H and various mDia1 mutants, followed by IP with anti flag antibody. (D) Schematic for mouse Phb2 truncation mutants. (E) Western blot to detect expression of Phb2 mutants. Lysates of HEK293T transfected with Phb2 mutants were analysed using anti-flag antibody. GAPDH was used as a loading control. (F) Co-IP of Phb2 mutants and mDia1 to map interaction domains. Lysates from HEK293T cells co-transfected with various Phb2 mutants and mDia1ΔN3 were subjected to IP using anti-flag antibody. Input lanes of Phb2 Amino, Central, Carboxy and 120-232 in the GFP blot, represent lower exposures cropped from the same blot and input lanes from Central, Carboxy and 120-232 in flag blot represent higher exposures cropped from the same blot. The numbers represent aa positions (A, D).

Article Snippet: Expression plasmids for GFP-tagged mouse mDia1, mDia1FL, mDia1ΔN3, mDia1F2, mDia1ΔN3(HindIII), mDia1H+P and mDia1CC were gifts from S Narumiya ( ). mDia1ΔN3-BD was cloned from mDia1ΔN3-pET28a into pGBKT7 (GAL4 binding domain vector-Clonetech) using Nde1 and BamH1.

Techniques: Western Blot, Expressing, Transfection, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Cytoplasmic sequestration of the RhoA effector mDiaphanous1 by Prohibitin2 promotes muscle differentiation

doi: 10.1101/283044

Figure Lengend Snippet: (A) Knockdown of mDia1 (but not Phb2) up-regulates MyoD and MyoG. qRT-PCR analysis of MyoD and MyoG transcripts in mDia1 and Phb2 knockdown in MT. C2C12 MB were transfected with control scrambled (SCR), mDia1, Phb2 or both mDia1+Phb2 siRNA pools and shifted to DM for 48 hours, followed by RNA extraction and qRT-PCR analysis. **p<0.01, ***p <0.001 compared with control SCR, n=3. Bar graph represents respective mRNA values normalized to GAPDH and SCR control. Error bars represent ± s.e.m. (B, C) Overexpressed mDia1ΔN3 represses MyoD and MyoG in MT, while co-expressed Phb2 reverses the repression. qRT-PCR analysis of MyoD (B) and MyoG (C) transcripts respectively in C2C12 transiently transfected with mDia1ΔN3 and Phb2 and shifted to DM for 36 hours. ***p<0.001, p<0.01, n=3. Bar graphs indicate normalised mRNA values. UT and GAPDH were used for normalisation of mRNA levels. Error bars represent ± s.e.m. (D, E) MyoG protein (but not MyoD) is repressed by mDia1ΔN3. Immunostaining of endogenous MyoD and MyoG in MT ectopically expressing mDia1 and Phb2. C2C12 were transfected with mDia1ΔN3 and Phb2-FL and shifted to DM for 36 hours, followed by IFA for Flag (Phb2), GFP (mDia1), MyoD and MyoG. Percentage of MyoD (D) and MyoG (E) positive nuclei were determined by counting atleast 200 cells. ***p<0.0001, n=3. (F) Representative images of endogenous MyoG protein during over-expression of mDia1ΔN3 and Phb2 during differentiation. Scale bar represents 20 μm. UT-Untransfected, FL-Full-length.

Article Snippet: Expression plasmids for GFP-tagged mouse mDia1, mDia1FL, mDia1ΔN3, mDia1F2, mDia1ΔN3(HindIII), mDia1H+P and mDia1CC were gifts from S Narumiya ( ). mDia1ΔN3-BD was cloned from mDia1ΔN3-pET28a into pGBKT7 (GAL4 binding domain vector-Clonetech) using Nde1 and BamH1.

Techniques: Quantitative RT-PCR, Transfection, RNA Extraction, Immunostaining, Expressing, Over Expression

Journal: bioRxiv

Article Title: Cytoplasmic sequestration of the RhoA effector mDiaphanous1 by Prohibitin2 promotes muscle differentiation

doi: 10.1101/283044

Figure Lengend Snippet: C2C12 were transfected with various mDia1 and Phb2 mutants along with MyoG-promoter reporter construct and shifted to DM for 72 hrs, followed by lysis and dual-luciferase assays. (A) Normalised MyoG promoter activity in MT transfected with mDia1ΔN3, mDia1H+P or Phb2-FL. *p<0.05, n=3. (B) Normalised MyoG promoter activity in MT transfected with mDia1ΔN3(HindIII), mDia1CC or Phb2-FL. *p<0.05, n=3. (C) Schematic illustrating FH2 motif (aa 946-1010)-mediated regulation of MyoG promoter by mDia1 mutants and Phb2. The squiggle represents the common domains not depicted. The FH2 motif is indicated by the stripped box within the dotted grey box representing the FH2 domain. (D) Normalised MyoG promoter activity in MT transfected with mDia1ΔN3, Phb2 carboxy or Phb2 amino. **p<0.01, n=3. For all Luciferase assays performed, Luciferase readings were normalised to Renilla Luciferase, empty pGL3 vector and basal DRR or MyoG promoter activity, to correct for background luminescence and transfection efficiency. Bar graphs represent normalised Luciferase values. Error bars represent ± s.e.m. FL-Full-length. UT-Untransfected.

Article Snippet: Expression plasmids for GFP-tagged mouse mDia1, mDia1FL, mDia1ΔN3, mDia1F2, mDia1ΔN3(HindIII), mDia1H+P and mDia1CC were gifts from S Narumiya ( ). mDia1ΔN3-BD was cloned from mDia1ΔN3-pET28a into pGBKT7 (GAL4 binding domain vector-Clonetech) using Nde1 and BamH1.

Techniques: Transfection, Construct, Lysis, Luciferase, Activity Assay, Plasmid Preparation