Journal: Frontiers in Endocrinology

Article Title: Regulation of Melanocortin-4 Receptor Pharmacology by Two Isoforms of Melanocortin Receptor Accessory Protein 2 in Topmouth Culter ( Culter alburnus )

doi: 10.3389/fendo.2020.00538

Figure Lengend Snippet: Nucleotide and deduced amino acid sequences and phylogenetic tree of caMRAP2a and caMRAP2b. (A) Nucleotide and deduced amino acid sequences of caMRAP2a. (B) Nucleotide and deduced amino acid sequences of caMRAP2b. The positions of nucleotide and amino acid sequences are indicated on both sides. Shaded box shows putative TMD. Open boxes denote putative N -linked glycosylation sites. Underline show initiation and stop codons. Asterisk (*) indicates stop codon. (C) Phylogenetic tree of MRAP2s. The tree was constructed by the NJ method. Numbers at nodes indicate the bootstrap value, as percentages, obtained for 1,000 replicates. Black triangle dot show caMRAP2a and caMRAP2b. MRAP2s: Culter alburnus (topmouth culter, MRAP2a: MT163516, and MRAP2b: MT163517), Danio rerio (zebrafish, MRAP2a: F8W4H9.1, and MRAP2b: F8W4H9.1), Epinephelus coioides (orange-spotted grouper, QED39647.1), Oryzias latipes (Japanese medaka, XP_023809099.1), Maylandia zebra (zebra mbuna, XP_004568825.1), Amphiprion ocellaris (anemonefish, XP_023122806.1), Kryptolebias marmoratus (mangrove rivulus, XP_017267334.1), Oncorhynchus mykiss (rainbow trout, XP_021467183.1), Oreochromis niloticus (Nile tilapia, XP_003458293.2), Gallus gallus (chicken, ALO81626.1), Ictidomys tridecemlineatus (thirteen-lined ground squirrel, XP_021581743.1), Oncorhynchus tshawytscha (Chinook salmon, XP_024278413.1), Mus caroli (Ryukyu mouse, XP_021029091.1), Pteropus alecto (black flying fox, XP_006926405.1), Puma concolor (puma, XP_025781535.1), Homo sapiens (human, AAH10003.2), Bubalus bubalis (water buffalo, XP_006054803.2), Macaca nemestrina (pig-tailed macaque, XP_011764298.1), Triplophysa tibetana (MRAP2a: KAA0703529.1 and MRAP2b: KAA0720858.1), Anabarilius grahami (Kanglang fish, MRAP2b ROJ29330.1), Carassius auratus (goldfish, MRAP2a: XP_026139519).

Article Snippet: N-terminal c-myc-tagged caMC4R, N-terminal FLAG-tagged caMRAP2a, and N-terminal FLAG-tagged caMRAP2b were synthesized and subcloned into pcDNA3.1 vector by GenScript (Piscataway, NJ, USA) to obtain the expression plasmids.

Techniques: Construct

Journal: Frontiers in Endocrinology

Article Title: Regulation of Melanocortin-4 Receptor Pharmacology by Two Isoforms of Melanocortin Receptor Accessory Protein 2 in Topmouth Culter ( Culter alburnus )

doi: 10.3389/fendo.2020.00538

Figure Lengend Snippet: Modulation of caMC4R expression by caMRAP2a or caMRAP2b. Cell surface (A,C) and total expression (B,D) of caMC4R was measured by flow cytometry. HEK293T cells were cotransfected with different ratios of caMC4R/caMRAP2a or caMC4R/caMRAP2b (1:0, 1:1, 1:3, and 1:5). The results were calculated as % of 1:0 group after correction of the background staining (fluorescence in cells transfected with empty vector, pcDNA3.1). Each data point represented the mean ± SEM ( n = 3–4). Different letters indicated significant difference ( P < 0.05) (One-way ANOVA followed by Tukey-test).

Article Snippet: N-terminal c-myc-tagged caMC4R, N-terminal FLAG-tagged caMRAP2a, and N-terminal FLAG-tagged caMRAP2b were synthesized and subcloned into pcDNA3.1 vector by GenScript (Piscataway, NJ, USA) to obtain the expression plasmids.

Techniques: Expressing, Flow Cytometry, Staining, Fluorescence, Transfection, Plasmid Preparation

Journal: Frontiers in Endocrinology

Article Title: Regulation of Melanocortin-4 Receptor Pharmacology by Two Isoforms of Melanocortin Receptor Accessory Protein 2 in Topmouth Culter ( Culter alburnus )

doi: 10.3389/fendo.2020.00538

Figure Lengend Snippet: Modulation of caMC4R pharmacology by caMRAP2a or caMRAP2b. Binding (A,B) and signaling properties (C,D) of caMC4R to α-MSH or ACTH (1–24) upon co-expression of caMRAP2a or caMRAP2b were measured. HEK293T cells were co-transfected with caMC4R/caMRAP2a or caMC4R/caMRAP2b in two different ratios (1:0, and 1:5). Binding and signaling properties were calculated as described in detail in , , respectively.

Article Snippet: N-terminal c-myc-tagged caMC4R, N-terminal FLAG-tagged caMRAP2a, and N-terminal FLAG-tagged caMRAP2b were synthesized and subcloned into pcDNA3.1 vector by GenScript (Piscataway, NJ, USA) to obtain the expression plasmids.

Techniques: Binding Assay, Expressing, Transfection

Journal: Frontiers in Endocrinology

Article Title: Regulation of Melanocortin-4 Receptor Pharmacology by Two Isoforms of Melanocortin Receptor Accessory Protein 2 in Topmouth Culter ( Culter alburnus )

doi: 10.3389/fendo.2020.00538

Figure Lengend Snippet: The effect of caMRAP2a or caMRAP2b on ligand binding properties of caMC4R.

Article Snippet: N-terminal c-myc-tagged caMC4R, N-terminal FLAG-tagged caMRAP2a, and N-terminal FLAG-tagged caMRAP2b were synthesized and subcloned into pcDNA3.1 vector by GenScript (Piscataway, NJ, USA) to obtain the expression plasmids.

Techniques: Ligand Binding Assay

Journal: Frontiers in Endocrinology

Article Title: Regulation of Melanocortin-4 Receptor Pharmacology by Two Isoforms of Melanocortin Receptor Accessory Protein 2 in Topmouth Culter ( Culter alburnus )

doi: 10.3389/fendo.2020.00538

Figure Lengend Snippet: The effect of caMRAP2a or caMRAP2b on cAMP signaling properties of caMC4R.

Article Snippet: N-terminal c-myc-tagged caMC4R, N-terminal FLAG-tagged caMRAP2a, and N-terminal FLAG-tagged caMRAP2b were synthesized and subcloned into pcDNA3.1 vector by GenScript (Piscataway, NJ, USA) to obtain the expression plasmids.

Techniques:

Journal: Frontiers in Endocrinology

Article Title: Regulation of Melanocortin-4 Receptor Pharmacology by Two Isoforms of Melanocortin Receptor Accessory Protein 2 in Topmouth Culter ( Culter alburnus )

doi: 10.3389/fendo.2020.00538

Figure Lengend Snippet: The effects of caMRAP2a or caMRAP2b on caMC4R signaling. HEK293T cells were co-transfected with different ratios of caMC4R/caMRAP2a or caMC4R/caMRAP2b (1:0, 1:1, 1:3, and 1:5) and cAMP levels under basal (A,B) or stimulated [with 10 −6 M α-MSH, (C,D) ] conditions were measured. Data are expressed as % of 1:0 group. Values are expressed as mean ± SEM ( n = 3). Different letters indicate significant difference (one-way ANOVA followed by Tukey-test).

Article Snippet: N-terminal c-myc-tagged caMC4R, N-terminal FLAG-tagged caMRAP2a, and N-terminal FLAG-tagged caMRAP2b were synthesized and subcloned into pcDNA3.1 vector by GenScript (Piscataway, NJ, USA) to obtain the expression plasmids.

Techniques: Transfection

Journal: Endocrine Connections

Article Title: Topmouth culter melanocortin-3 receptor: regulation by two isoforms of melanocortin-2 receptor accessory protein 2

doi: 10.1530/EC-21-0459

Figure Lengend Snippet: Nucleotide and deduced amino acid sequences (A) and phylogenetic tree (B) of caMC3R. Positions of nucleotide and amino acid sequences are indicated on both sides. N-linked glycosylation sites are present in open boxes. Shaded boxes show putative TMD1-7. Oval frame denotes potential phosphorylation site. The conserved motifs (PMY, DRY, and DPxxY) are underlined. Asterisk (*) shows stop codon. The tree was constructed by the neighbor-joining ethod. Numbers at nodes indicate the bootstrap value, as percentages, obtained for 1000 replicates. Black dot denotes culter MC3R. MC3Rs: Culteralburnus (topmouth culter, MW419813), Megalobramaamblycephala (Wuchang bream, AWA81517.1), Carassiusauratus (goldfish, BAJ83473.1), Daniorerio (zebrafish, AAO24744.1), Oncorhynchuskisutch (coho salmon, XP_020360426.1), Homo sapiens (human, NP_063941.3), Musmusculus (mouse, AAI03670.1), Gallus gallus (chicken, XP_004947293.1), Susscrofa (pig, AFK25142.1), Rattusnorvegicus (rat, NP_001020441.3), Equuscaballus (horse, NP_001243901.1), Pangasianodonhypophthalmus (iridescent shark, XP_026770221.1), Astyanaxmexicanus (Mexican tetra, XP_007231215.1), Oryx gazella (gemsbok, AFH58734.1), Pteropusvampyrus (large flying fox, XP_011368476.1), Pteropusalecto (black flying fox, XP_006921991.1), Dasypusnovemcinctus (nine-banded armadillo, XP_004447768.1), Feliscatus (cat, XP_023106851.1), Loxodontaafricana (African bush elephant, XP_003419952.1), Oncorhynchustshawytscha (chinook salmon, XP_024229914.1), Salvelinusalpinus (Arctic char, XP_023994975.1), Amazonaaestiva (turquoise-fronted amazon, KQL61336.1), Scleropagesformosus (Asian arowana, XP_018615783.1), Pelodiscussinensis (Chinese softshell turtle, XP_006129463.1), Terrapenecarolinatriunguis (common box turtle, XP_024059166.1), and Alligator sinensis (Chinese alligator, XP_006018246.1).

Article Snippet: N-terminal myc-tagged caMC3R was commercially synthesized and subcloned into pcDNA3.1 by GenScript to generate the plasmid used for transfection.

Techniques: Construct

Journal: Endocrine Connections

Article Title: Topmouth culter melanocortin-3 receptor: regulation by two isoforms of melanocortin-2 receptor accessory protein 2

doi: 10.1530/EC-21-0459

Figure Lengend Snippet: Ligand binding properties of caMC3R. Different concentrations of unlabeled NDP-MSH (A), α-MSH (B), des-α-MSH (C), and ACTH (1–24) (D) were used to displace the binding of 125 I-NDP-MSH. Results are expressed as % of hMC3R binding ± range from duplicate determinations within one experiment. All experiments were repeated at least three independent times.

Article Snippet: N-terminal myc-tagged caMC3R was commercially synthesized and subcloned into pcDNA3.1 by GenScript to generate the plasmid used for transfection.

Techniques: Ligand Binding Assay, Binding Assay

Journal: Endocrine Connections

Article Title: Topmouth culter melanocortin-3 receptor: regulation by two isoforms of melanocortin-2 receptor accessory protein 2

doi: 10.1530/EC-21-0459

Figure Lengend Snippet: The ligand binding properties of caMC3R.

Article Snippet: N-terminal myc-tagged caMC3R was commercially synthesized and subcloned into pcDNA3.1 by GenScript to generate the plasmid used for transfection.

Techniques: Ligand Binding Assay

Journal: Endocrine Connections

Article Title: Topmouth culter melanocortin-3 receptor: regulation by two isoforms of melanocortin-2 receptor accessory protein 2

doi: 10.1530/EC-21-0459

Figure Lengend Snippet: Signaling properties of caMC3R. HEK293T cells were transiently transfected with hMC3R or caMC3R plasmids. Different concentrations of NDP-MSH (A), α-MSH (B), des-α-MSH (C), and ACTH (1–24) (D) were used to stimulate the cells. (E) Constitutive activities of caMC3R in cAMP pathway. Increasing concentrations of caMC3R plasmid were transfected into HEK293T cells. Cells transfected with empty pcDNA3.1 vector were used as the control group. Data are mean ± s.e.m. from triplicate measurements within one experiment. All experiments were performed at least three times independently.

Article Snippet: N-terminal myc-tagged caMC3R was commercially synthesized and subcloned into pcDNA3.1 by GenScript to generate the plasmid used for transfection.

Techniques: Transfection, Plasmid Preparation

Journal: Endocrine Connections

Article Title: Topmouth culter melanocortin-3 receptor: regulation by two isoforms of melanocortin-2 receptor accessory protein 2

doi: 10.1530/EC-21-0459

Figure Lengend Snippet: The signaling properties of caMC3R.

Article Snippet: N-terminal myc-tagged caMC3R was commercially synthesized and subcloned into pcDNA3.1 by GenScript to generate the plasmid used for transfection.

Techniques:

Journal: Endocrine Connections

Article Title: Topmouth culter melanocortin-3 receptor: regulation by two isoforms of melanocortin-2 receptor accessory protein 2

doi: 10.1530/EC-21-0459

Figure Lengend Snippet: Regulation of caMC3R expression and signaling by caMRAP2a or caMRAP2b. Cell surface expression (A) and total expression (B) of caMC3R modulated by caMRAP2a or caMRAP2b were measured by flow cytometry. Basal (C) and maximal signaling (D) of caMC3R regulated by MRAP2a or MRAP2b were determined by RIA. HEK293T cells were co-transfected with different ratios of caMC3R/caMRAP2a or caMC3R/caMRAP2b (1:0, 1:1, 1:3, and 1:5). The empty vector pcDNA3.1 fluorescence was used for background staining. The results are calculated as % of 1:0 group. Each data point represented as the mean ± s.e.m. ( n = 3 – 4). Different letters indicate significant difference ( P < 0.05) (one-way ANOVA followed by Tukey test).

Article Snippet: N-terminal myc-tagged caMC3R was commercially synthesized and subcloned into pcDNA3.1 by GenScript to generate the plasmid used for transfection.

Techniques: Expressing, Flow Cytometry, Transfection, Plasmid Preparation, Fluorescence, Staining

Journal: Endocrine Connections

Article Title: Topmouth culter melanocortin-3 receptor: regulation by two isoforms of melanocortin-2 receptor accessory protein 2

doi: 10.1530/EC-21-0459

Figure Lengend Snippet: Modulation of caMC3R pharmacology by caMRAP2a or caMRAP2b. Ligand binding (A, B) and signaling (C, D) properties of caMC3R to α-MSH or ACTH (1–24) upon co-expression of caMC3R with caMRAP2a or caMRAP2b were measured. HEK293T cells were co-transfected with caMC3R/caMRAP2a or caMC3R/caMRAP2b in two different ratios (1:0 and 1:5). Results of binding properties were calculated as % of hMC3R binding ± range from duplicate determinations within one experiment. All experiments were measured at least three times independently.

Article Snippet: N-terminal myc-tagged caMC3R was commercially synthesized and subcloned into pcDNA3.1 by GenScript to generate the plasmid used for transfection.

Techniques: Ligand Binding Assay, Expressing, Transfection, Binding Assay

Journal: Endocrine Connections

Article Title: Topmouth culter melanocortin-3 receptor: regulation by two isoforms of melanocortin-2 receptor accessory protein 2

doi: 10.1530/EC-21-0459

Figure Lengend Snippet: The effect of caMRAP2a or caMRAP2b on ligand binding properties of caMC3R.

Article Snippet: N-terminal myc-tagged caMC3R was commercially synthesized and subcloned into pcDNA3.1 by GenScript to generate the plasmid used for transfection.

Techniques: Ligand Binding Assay

Journal: Endocrine Connections

Article Title: Topmouth culter melanocortin-3 receptor: regulation by two isoforms of melanocortin-2 receptor accessory protein 2

doi: 10.1530/EC-21-0459

Figure Lengend Snippet: The effect of caMRAP2a or caMRAP2b on cAMP signaling of caMC3R.

Article Snippet: N-terminal myc-tagged caMC3R was commercially synthesized and subcloned into pcDNA3.1 by GenScript to generate the plasmid used for transfection.

Techniques:

Journal: Nature Communications

Article Title: Proton-coupled transport mechanism of the efflux pump NorA

doi: 10.1038/s41467-024-48759-3

Figure Lengend Snippet: a Structures of NorA at pH 5.0 (left) and NorA E222Q,D307N at pH 7.5 (right) determined in complex with FabDA1. The structure at pH 5.0 corresponds to NorA protonated at Glu222 and Asp307. NorA TM helices are colored in rainbow, and CDRH3 of FabDA1 is displayed in grey. b Left: surface representations of NTD corresponding to TM1 to TM6 (in spring green) and CTD corresponding to TM7 to TM12 (in light grey). The two proton binding positions at Glu222 and Asp307 within the CTD are schematically depicted. Right: surface representations of TM5 (spring green) packing against the CTD (in light grey). c Expanded view of interactions between TM5 (in light grey) and Glu222 and Asp307 within the CTD as depicted in a partially transparent surface with select residues displayed in sticks. The surface representation is colored as follows: hydrophobic residues in yellow, anionic residues in red, cationic residues in blue, and all other residues in light grey . d Surface and cutaway views of the electrostatic potential plotted on NorA. The sites of Glu222 and Asp307 are indicated in NorA. Blue, red, and white colors correspond to positively, negatively, and neutrally charged sites, respectively.

Article Snippet: Primary antibodies specific to the C-terminal Myc tag on NorA (Genscript) and SrtA were used, with dilutions of 1:1000 and 1:20,000 for NorA and SrtA , respectively.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Proton-coupled transport mechanism of the efflux pump NorA

doi: 10.1038/s41467-024-48759-3

Figure Lengend Snippet: a , b Left: cryo-EM structures of NorA D307N ( a ) and NorA E222Q ( b ) determined at pH 7.5 in complex with FabDA1 (in spring green) superimposed with the NorA structure determined at pH 5.0 (in grey). Right: same superimposition as in the left panel, except with TM5 highlighted (in spring green) and the structure rotated by 180°. Other helices are colored in white and made partially transparent. c – f Nearest distances between Glu222 or Gln222 in TM7 (in green) to the backbone carbonyl oxygen of Phe140 in TM5 (in spring green) and Asp307 or Asn307 in TM10 (in gold) to Asn137 in TM5 for NorA ( c ), NorA E222Q,D307N ( d ), NorA D307N ( e ), and NorA E222Q ( f ). Dashed black lines correspond to the indicated distances (in Å).

Article Snippet: Primary antibodies specific to the C-terminal Myc tag on NorA (Genscript) and SrtA were used, with dilutions of 1:1000 and 1:20,000 for NorA and SrtA , respectively.

Techniques: Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Proton-coupled transport mechanism of the efflux pump NorA

doi: 10.1038/s41467-024-48759-3

Figure Lengend Snippet: a Cytoplasmic views of the NorA structure determined at pH 5.0 using cryo-EM (left) and NorA deprotonated at Glu222 and Asp307 derived from MD simulations (right). TM domain helices are numbered for reference, and TM5 (in spring green) and TM10 (in gold) are highlighted among other helices (in grey). The thick, red line corresponds to the indicated distance (in Å) between the Cα atoms of Ala126 in TM5 and Ser318 in TM10, illustrating the separation of TM5 from the CTD. b The water radial distribution function (RDF) surrounding Glu222 (left) or Asp307 (right) calculated from NorA MD simulations where Glu222 and Asp307 were protonated (blue) or deprotonated (magenta). c Representative snapshots from MD simulations of the water distribution in the substrate binding pocket of NorA protonated or deprotonated at Glu222 and Asp307. Water molecules are represented by red (oxygens) and white (hydrogens) spheres, Glu222 and Asp307 are highlighted by spring green and yellow surfaces, and TM helices are colored in light grey.

Article Snippet: Primary antibodies specific to the C-terminal Myc tag on NorA (Genscript) and SrtA were used, with dilutions of 1:1000 and 1:20,000 for NorA and SrtA , respectively.

Techniques: Cryo-EM Sample Prep, Derivative Assay, Binding Assay

Journal: Nature Communications

Article Title: Proton-coupled transport mechanism of the efflux pump NorA

doi: 10.1038/s41467-024-48759-3

Figure Lengend Snippet: A schematic depiction of the “belt and suspenders” mechanism. The models show surface representations of inward-occluded cryo-EM structures of wild-type NorA at pH 5.0, NorA E222Q at pH 7.5, and NorA D307N at pH 7.5 and the inward-open model predicted from AlphaFold2 (likely to be apo based on MD simulations). Red lines connecting the NTD and CTD denote the “belt” or “suspenders”. “E222 − ” and “D307 − ” correspond to deprotonated Glu222 and Asp307 residues and “E222” and “D307” correspond to protonated Glu222 and Asp307 residues. The NTD and CTD are colored in blue and orange, respectively.

Article Snippet: Primary antibodies specific to the C-terminal Myc tag on NorA (Genscript) and SrtA were used, with dilutions of 1:1000 and 1:20,000 for NorA and SrtA , respectively.

Techniques: Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Proton-coupled transport mechanism of the efflux pump NorA

doi: 10.1038/s41467-024-48759-3

Figure Lengend Snippet: a Diagram of NorA transport stoichiometries of 2:1 H + :ethidium (electrogenic, top) or 1:1 H + :ethidium (electroneutral, bottom) and the two energy sources for efflux: proton gradient (∆pH) and membrane potential (∆ψ). Electroneutral transport is driven by ∆pH, while electrogenic transport can be driven by ∆pH or ∆ψ. b Growth inhibition experiments at pH 7.8 of MRSA ∆ norA transformed with a hemin-inducible plasmid containing the wild-type norA gene (black circles) or a dead norA mutant control (grey triangles) in the presence of variable ethidium bromide concentration (0 to 125 μg/mL). Bacterial growth is displayed by the OD 600nm value at the 12 hr timepoint. Solid lines correspond to non-linear fits of experimental data to determine the IC 50 values. The standard deviations are derived from four replicates obtained from two independent experiments. c IC 50 values of plasmids containing wild-type norA divided by the respective control plasmid as a function of different external pH values ranging from 6.5 to 8.5. Mean values ± s.d. are depicted for the IC 50 values. d Proposed NorA transport cycle. The NTD and CTD are colored in blue and orange, “S” is the drug substrate, “E222 − ” and “D307 − ” represents deprotonated Glu222 and Asp307, and “E222” and “D307” are protonated Glu222 and Asp307. Step 1: Deprotonation of Glu222 and Asp307 in the inward-occluded state releases the TM5 tether, leading to opening of the substrate binding pocket. Step 2: drug binds to the inward-open conformation of NorA from the cytoplasmic side of the membrane. Step 3: conformational exchange flips the drug-bound inward-open state to the outward-open state. Step 4: drug is released into the periplasm, resulting in the apo, outward-open conformation. Step 5: Glu222 and Asp307 bind protons in the periplasm to give an outward-occluded conformation. Step 6: NorA undergoes conformational exchange to the inward-occluded conformation, preparing it for the next round of substrate turnover.

Article Snippet: Primary antibodies specific to the C-terminal Myc tag on NorA (Genscript) and SrtA were used, with dilutions of 1:1000 and 1:20,000 for NorA and SrtA , respectively.

Techniques: Membrane, Inhibition, Transformation Assay, Plasmid Preparation, Mutagenesis, Control, Concentration Assay, Derivative Assay, Binding Assay

Journal: Nature Communications

Article Title: Proton-coupled transport mechanism of the efflux pump NorA

doi: 10.1038/s41467-024-48759-3

Figure Lengend Snippet: a View of the inward-occluded NorA cryo-EM structure at pH 5.0 displaying Glu222 and Asp307 and residues within 5 Å from these two residues. b Norfloxacin minimum inhibitory concentration (MIC) values were measured in an MRSA strain where the native norA gene was disrupted by a transposon insertion (MRSA Δ norA ). Wild-type NorA and indicated mutants were encoded on a hemin-inducible plasmid. The number of independent MIC experiments (five for empty vector Δ norA , two for other variants) is represented by the black circles overlaid on the bar graph for each sample. Data are presented as mean values ± s.d. across independent experiments. The upper limit of MIC detection was set at 256 μg/mL. Statistical significance was assessed using an unpaired, two-tailed t-test for each single-site mutant in comparison to MRSA Δ norA + NorA. P values were labeled on each bar. c Immunoblotting results performed on MRSA cell lysates following the induction of MRSA Δ norA or MRSA Δ norA transformed with a hemin-inducible plasmid carrying wild-type NorA and NorA mutants. The immunoblots probed for the Myc tag on the C-terminus of NorA and SrtA (control protein in S. aureus ). Similar results were observed in two independent experiments. d Ethidium efflux assay results for MRSA Δ norA transformed with plasmids corresponding to empty vector (Δ norA ), wild-type NorA, E222Q, and D307N. The graph displays the normalized ethidium fluorescence (F/F0) over time, starting from the addition of glucose at time zero. Solid and shaded lines in the same color depict the mean values ± s.d. from two independent experiments with a total of four technical replicates. e A plot of the normalized fluorescence value (F/F0) at 1200 s from the data in d . The number of technical replicates from two independent experiments is represented by the black circles overlaid on the bar graph for each sample. Data are presented as mean values ± s.d. from four technical replicates. Statistical significance was evaluated using an unpaired, two-tailed t-test for individual single-site mutants compared to MRSA Δ norA + NorA. P values were labeled on each bar.

Article Snippet: Primary antibodies specific to the C-terminal Myc tag on NorA (Genscript) and SrtA were used, with dilutions of 1:1000 and 1:20,000 for NorA and SrtA , respectively.

Techniques: Cryo-EM Sample Prep, Concentration Assay, Plasmid Preparation, Two Tailed Test, Mutagenesis, Comparison, Labeling, Western Blot, Transformation Assay, Control, Fluorescence

Journal: PLoS Genetics

Article Title: Mapping the degradation pathway of a disease-linked aspartoacylase variant

doi: 10.1371/journal.pgen.1009539

Figure Lengend Snippet: (A) U2OS cells were transiently transfected with the indicated constructs and a GFP expression plasmid to normalize for differences in transfection efficiency. ASPA and GFP protein levels were visualized by Western blotting. Vinculin was used as a loading control. (B) Western blot showing ASPA protein levels in U2OS cells transiently transfected with the indicated constructs prior to treatment with the translation inhibitor cycloheximide (CHX) for 12 hours (+) or the solvent DMSO as a control (-). Vinculin serves as a loading control. (C) U2OS cells were transiently transfected with the indicated constructs prior to treatment with the proteasome inhibitor bortezomib (BZ) for 8 hours (+) or, as a control, the solvent DMSO (-). ASPA protein levels were examined by Western blotting. Blotting for vinculin is used as a loading control. (D) Transiently transfected U2OS cells were treated with the Hsp70 inhibitor YM-01 for 24 hours (+) or the solvent DMSO (-) prior to protein extraction and Western blotting. Vinculin is used as a loading control. (E) U2OS cells were reverse transfected with the shown siRNAs, followed by transient transfection with the constructs shown at the top. Then, ASPA protein levels were examined by Western blotting. Vinculin serves as a loading control. (F) The soluble (S) and insoluble (P) fractions of protein were separated in cell extracts treated as in E prior to Western blotting. GAPDH and the Na/K ATPase serve as a loading control for the soluble and insoluble protein fraction, respectively. (G) U2OS cells were transiently transfected with HSPH1-Myc prior to Myc immunoprecipitation followed by Western blotting. (H) U2OS cells were reverse transfected with control siRNA (-) or siRNA targeting HSPH1 (+) prior to transfection of HA-ubiquitin and wild-type ASPA or C152W as indicated. The cells were used for denaturing HA immunoprecipitation followed by Western blotting. (I) U2OS cells were transiently co-transfected with Hsp104 and the indicated constructs, and ASPA protein levels were examined by Western blotting. Blotting for vinculin is used as a loading control. (J) Cells were treated as in I followed by differential centrifugation to separate soluble (S) and insoluble (P) protein fractions and Western blotting. Blotting for GAPDH and the Na/K ATPase is used as a loading control for the soluble and the insoluble fractions respectively.

Article Snippet: Human codon-optimized HSPH1 fused C-terminally to a Myc-tag was expressed from pcDNA3.1 (Genscript). cDNA encoding wild-type human ubiquitin containing an N-terminal HA-tag was expressed from pRK5-HA (Addgene).

Techniques: Transfection, Construct, Expressing, Plasmid Preparation, Western Blot, Protein Extraction, Immunoprecipitation, Centrifugation