Structured Review

Millipore monoclonal anti flag m2 antibody
Tests of guide strand 5’ chemical groups and nucleotides in AGO4 loading and slicing A . Cartoon illustrating alternative features of precursors and diced siRNA duplexes. B . Test of AGO4 loading for 24 nt guide RNAs with 5’ monophosphate (P), hydroxyl (OH), or triphosphate (PPP) groups. Input samples are shown in lanes 1-4. RNAs that co-IP with AGO4 are in lanes 5-8. RNAs were subjected to denaturing PAGE, transferred to membranes, and detected by RNA blot hybridization. Controls in lanes 1 and 5 lacked RNA. C . Target RNA slicing programmed by guide siRNAs with different 5’ end modifications. 24 nt guide RNAs with different 5’ groups, paired with a 5’ end-labeled 23 nt target RNA, are shown at the top. The predicted slice site is denoted by a black triangle. The alternative guide RNAs were incubated with or without AGO4 followed by incubation with 32 P-labeled 23 nt target RNA. RNAs were then subjected to denaturing PAGE. An end-labeled 12 nt size marker was run in lane 1. Lane 5 is a control in which AGO4 was included but guide RNA was omitted. D . Test for preferential loading of guide RNAs with different 5’ nucleotides. 24 nt RNAs with each of the four possible nucleotides at their 5’ ends were 32 P-end-labeled and incubated with <t>FLAG-tagged</t> recombinant AGO4. RNAs associated with AGO4 captured on <t>anti-FLAG</t> Dynabeads were resolved by denaturing PAGE (lanes 9-12). In mock IP control reactions (lanes 5-8), AGO4 was omitted. Lanes 1 to 4 show input RNA. See also Supplementary Figure S7 . E . Test of target RNA slicing programmed by guide RNAs with different 5’ nucleotides. The different guide RNAs were incubated with AGO4 (lanes 6 to 9) or without AGO4 (lanes 2 to 5) then incubated with end-labeled 23 nt target RNA. RNAs were then purified, subjected to denaturing PAGE and autoradiography.
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Images

1) Product Images from "Mechanisms of AGO4 slicing-enhanced RNA-directed DNA methylation"

Article Title: Mechanisms of AGO4 slicing-enhanced RNA-directed DNA methylation

Journal: bioRxiv

doi: 10.1101/2022.10.06.511223

Tests of guide strand 5’ chemical groups and nucleotides in AGO4 loading and slicing A . Cartoon illustrating alternative features of precursors and diced siRNA duplexes. B . Test of AGO4 loading for 24 nt guide RNAs with 5’ monophosphate (P), hydroxyl (OH), or triphosphate (PPP) groups. Input samples are shown in lanes 1-4. RNAs that co-IP with AGO4 are in lanes 5-8. RNAs were subjected to denaturing PAGE, transferred to membranes, and detected by RNA blot hybridization. Controls in lanes 1 and 5 lacked RNA. C . Target RNA slicing programmed by guide siRNAs with different 5’ end modifications. 24 nt guide RNAs with different 5’ groups, paired with a 5’ end-labeled 23 nt target RNA, are shown at the top. The predicted slice site is denoted by a black triangle. The alternative guide RNAs were incubated with or without AGO4 followed by incubation with 32 P-labeled 23 nt target RNA. RNAs were then subjected to denaturing PAGE. An end-labeled 12 nt size marker was run in lane 1. Lane 5 is a control in which AGO4 was included but guide RNA was omitted. D . Test for preferential loading of guide RNAs with different 5’ nucleotides. 24 nt RNAs with each of the four possible nucleotides at their 5’ ends were 32 P-end-labeled and incubated with FLAG-tagged recombinant AGO4. RNAs associated with AGO4 captured on anti-FLAG Dynabeads were resolved by denaturing PAGE (lanes 9-12). In mock IP control reactions (lanes 5-8), AGO4 was omitted. Lanes 1 to 4 show input RNA. See also Supplementary Figure S7 . E . Test of target RNA slicing programmed by guide RNAs with different 5’ nucleotides. The different guide RNAs were incubated with AGO4 (lanes 6 to 9) or without AGO4 (lanes 2 to 5) then incubated with end-labeled 23 nt target RNA. RNAs were then purified, subjected to denaturing PAGE and autoradiography.
Figure Legend Snippet: Tests of guide strand 5’ chemical groups and nucleotides in AGO4 loading and slicing A . Cartoon illustrating alternative features of precursors and diced siRNA duplexes. B . Test of AGO4 loading for 24 nt guide RNAs with 5’ monophosphate (P), hydroxyl (OH), or triphosphate (PPP) groups. Input samples are shown in lanes 1-4. RNAs that co-IP with AGO4 are in lanes 5-8. RNAs were subjected to denaturing PAGE, transferred to membranes, and detected by RNA blot hybridization. Controls in lanes 1 and 5 lacked RNA. C . Target RNA slicing programmed by guide siRNAs with different 5’ end modifications. 24 nt guide RNAs with different 5’ groups, paired with a 5’ end-labeled 23 nt target RNA, are shown at the top. The predicted slice site is denoted by a black triangle. The alternative guide RNAs were incubated with or without AGO4 followed by incubation with 32 P-labeled 23 nt target RNA. RNAs were then subjected to denaturing PAGE. An end-labeled 12 nt size marker was run in lane 1. Lane 5 is a control in which AGO4 was included but guide RNA was omitted. D . Test for preferential loading of guide RNAs with different 5’ nucleotides. 24 nt RNAs with each of the four possible nucleotides at their 5’ ends were 32 P-end-labeled and incubated with FLAG-tagged recombinant AGO4. RNAs associated with AGO4 captured on anti-FLAG Dynabeads were resolved by denaturing PAGE (lanes 9-12). In mock IP control reactions (lanes 5-8), AGO4 was omitted. Lanes 1 to 4 show input RNA. See also Supplementary Figure S7 . E . Test of target RNA slicing programmed by guide RNAs with different 5’ nucleotides. The different guide RNAs were incubated with AGO4 (lanes 6 to 9) or without AGO4 (lanes 2 to 5) then incubated with end-labeled 23 nt target RNA. RNAs were then purified, subjected to denaturing PAGE and autoradiography.

Techniques Used: Co-Immunoprecipitation Assay, Polyacrylamide Gel Electrophoresis, Northern blot, Hybridization, Labeling, Incubation, Marker, Recombinant, Purification, Autoradiography

Evidence that 23 nt siRNAs function as passenger strands for 24 nt guide RNAs. A . A simplified model for RNA-directed DNA methylation highlighting questions addressed in our study. B . Relative abundance of small RNA size classes at 24 nt siRNA-dominated loci in diverse species ( Lunardon et al., 2020 ). C . Transgene-encoded AGO4 proteins expressed from the native promoter in ago4-4 null mutant plants (ecotype Ws). D . Comparison of AGO4 and AGO4-SD levels IPed from inflorescence tissue. Duplicate gels were stained with Coomassie Brilliant Blue (top panel) or subjected to immunoblotting using anti-FLAG antibody (bottom panel). Non-transgenic Ws serves as a control in lane 1. E . Cartoon depicting AGO4 affinity capture and analysis of associated RNAs by 32 P end-labeling or deep sequencing. F . Autoradiogram of 5’ end-labeled RNAs in anti-FLAG IP fractions from non-transgenic plants (lane 1), plants expressing FLAG-AGO4 (lane 2) or plants expressing FLAG-AGO4-SD (lane 3).
Figure Legend Snippet: Evidence that 23 nt siRNAs function as passenger strands for 24 nt guide RNAs. A . A simplified model for RNA-directed DNA methylation highlighting questions addressed in our study. B . Relative abundance of small RNA size classes at 24 nt siRNA-dominated loci in diverse species ( Lunardon et al., 2020 ). C . Transgene-encoded AGO4 proteins expressed from the native promoter in ago4-4 null mutant plants (ecotype Ws). D . Comparison of AGO4 and AGO4-SD levels IPed from inflorescence tissue. Duplicate gels were stained with Coomassie Brilliant Blue (top panel) or subjected to immunoblotting using anti-FLAG antibody (bottom panel). Non-transgenic Ws serves as a control in lane 1. E . Cartoon depicting AGO4 affinity capture and analysis of associated RNAs by 32 P end-labeling or deep sequencing. F . Autoradiogram of 5’ end-labeled RNAs in anti-FLAG IP fractions from non-transgenic plants (lane 1), plants expressing FLAG-AGO4 (lane 2) or plants expressing FLAG-AGO4-SD (lane 3).

Techniques Used: DNA Methylation Assay, Mutagenesis, Staining, Transgenic Assay, End Labeling, Sequencing, Labeling, Expressing

AGO4 retains sliced target RNAs in vitro and in vivo A . Cartoon of the strategy for testing AGO4 release or retention of sliced target RNAs. B . Test for retention of 23 nt target strand 5’ cleavage products. At the top are sequences of a 24 nt guide RNA with 100% complementarity to the 32 P-labeled target RNA s and a guide RNA with three nucleotides of non-complementarity (highlighted in green) to the target. In the experiment, FLAG-AGO4 immobilized on anti-FLAG Dynabeads was incubated with buffer only (lanes 2 and 3), with the 24 nt guide RNA having perfect complementarity to the target RNA (lanes 4 and 5), or with 24 nt guide RNAs with mismatches to the target RNA (lanes 6 and 7). RNAs in the supernatant (S) and washed bead (B) fractions were purified, subjected to denaturing PAGE and autoradiography. Lane 1 is an anti-FLAG IP control using non-transgenic plants. C . Test for retention of 5’ cleavage products of a 51 nt RNA target. The experiment was conducted as for panel B. D . Both 5’ and 3’ cleavage products of long RNAs are retained by AGO4 in vitro . The experiment was conducted as for panel B except that a body-labeled 54 nt target RNA was used. The expected sizes of its slicing products are 22 and 32 nt. E . Sliced products of RdDM target locus RNAs are retained by AGO4 in vivo . The cartoon shows how 24 nt guide RNAs of variable sequence (orange) would pair with complementary target RNAs. 5’ and 3’ cleavage products are color-coded grey and green respectively. At position 10 of the 3’ slicing products (green fragments), a strong uridine (U) signature is expected due to complementarity to the adenosine (A) at the guide strand 5’ end. The sequence logos below show RNA-seq analyses for RNAs longer than 30 nt that co-IPed with wild-type AGO4 or AGO4-SD. The arrow denotes the position of the expected U signature.
Figure Legend Snippet: AGO4 retains sliced target RNAs in vitro and in vivo A . Cartoon of the strategy for testing AGO4 release or retention of sliced target RNAs. B . Test for retention of 23 nt target strand 5’ cleavage products. At the top are sequences of a 24 nt guide RNA with 100% complementarity to the 32 P-labeled target RNA s and a guide RNA with three nucleotides of non-complementarity (highlighted in green) to the target. In the experiment, FLAG-AGO4 immobilized on anti-FLAG Dynabeads was incubated with buffer only (lanes 2 and 3), with the 24 nt guide RNA having perfect complementarity to the target RNA (lanes 4 and 5), or with 24 nt guide RNAs with mismatches to the target RNA (lanes 6 and 7). RNAs in the supernatant (S) and washed bead (B) fractions were purified, subjected to denaturing PAGE and autoradiography. Lane 1 is an anti-FLAG IP control using non-transgenic plants. C . Test for retention of 5’ cleavage products of a 51 nt RNA target. The experiment was conducted as for panel B. D . Both 5’ and 3’ cleavage products of long RNAs are retained by AGO4 in vitro . The experiment was conducted as for panel B except that a body-labeled 54 nt target RNA was used. The expected sizes of its slicing products are 22 and 32 nt. E . Sliced products of RdDM target locus RNAs are retained by AGO4 in vivo . The cartoon shows how 24 nt guide RNAs of variable sequence (orange) would pair with complementary target RNAs. 5’ and 3’ cleavage products are color-coded grey and green respectively. At position 10 of the 3’ slicing products (green fragments), a strong uridine (U) signature is expected due to complementarity to the adenosine (A) at the guide strand 5’ end. The sequence logos below show RNA-seq analyses for RNAs longer than 30 nt that co-IPed with wild-type AGO4 or AGO4-SD. The arrow denotes the position of the expected U signature.

Techniques Used: In Vitro, In Vivo, Labeling, Incubation, Purification, Polyacrylamide Gel Electrophoresis, Autoradiography, Transgenic Assay, Sequencing, RNA Sequencing Assay

2) Product Images from "Computational Modeling of the Chlamydial Developmental Cycle Reveals a Potential Role for Asymmetric Division"

Article Title: Computational Modeling of the Chlamydial Developmental Cycle Reveals a Potential Role for Asymmetric Division

Journal: bioRxiv

doi: 10.1101/2022.09.01.506140

IBs mature directly into EBs. Cos-7 cells were infected with L2-E- ftsI 3XFLAG-BMAMEO. Infected cells were treated at 18 hpi with either vehicle (UNT), ciprofloxacin (CIP), or induced for FtsI3XFLAG expression (FtsI). Samples were fixed at 22 hpi or 34 hpi, stained with DAPI and an anti-FLAG antibody. Images are z-projections from confocal micrographs showing hctA prom-mEos3.2: green, hctB prom-mKate2: red, DAPI: cyan, anti-FLAG: magenta. A . Representative confocal micrographs of 22 hpi cells along with quantification of hctA prom-mEos3.2 and hctB prom-mKate2 expression levels at the single cell level. Quantification of DAPI positive cells was performed using Trackmate from 3 individual inclusions per treatment per time point. Each color corresponds to chlamydial cells quantified within the same inclusion. B . Confocal micrographs of L2-E- ftsI 3XFLAG-BMAMEO infected cells at 34 hpi either treated with vehicle (UNT), CIP, or induced to express FtsI. Inserts demonstrate the overlap of hctA prom-mEos3.2 and hctB prom-mKate2 within single cells. Quantification of DAPI positive cells was performed using Trackmate from 3 individual inclusions per treatment per time point. Each color corresponds to chlamydial cells quantified within the same inclusion. Scale bar = 10 µm.
Figure Legend Snippet: IBs mature directly into EBs. Cos-7 cells were infected with L2-E- ftsI 3XFLAG-BMAMEO. Infected cells were treated at 18 hpi with either vehicle (UNT), ciprofloxacin (CIP), or induced for FtsI3XFLAG expression (FtsI). Samples were fixed at 22 hpi or 34 hpi, stained with DAPI and an anti-FLAG antibody. Images are z-projections from confocal micrographs showing hctA prom-mEos3.2: green, hctB prom-mKate2: red, DAPI: cyan, anti-FLAG: magenta. A . Representative confocal micrographs of 22 hpi cells along with quantification of hctA prom-mEos3.2 and hctB prom-mKate2 expression levels at the single cell level. Quantification of DAPI positive cells was performed using Trackmate from 3 individual inclusions per treatment per time point. Each color corresponds to chlamydial cells quantified within the same inclusion. B . Confocal micrographs of L2-E- ftsI 3XFLAG-BMAMEO infected cells at 34 hpi either treated with vehicle (UNT), CIP, or induced to express FtsI. Inserts demonstrate the overlap of hctA prom-mEos3.2 and hctB prom-mKate2 within single cells. Quantification of DAPI positive cells was performed using Trackmate from 3 individual inclusions per treatment per time point. Each color corresponds to chlamydial cells quantified within the same inclusion. Scale bar = 10 µm.

Techniques Used: Infection, Expressing, Staining

IB to EB development is replication independent. Cos-7 cells were infected with either purified L2-BMELVA or L2-E- ftsI 3XFLAG-BMELVA. L2-BMELVA infected cells were treated with either vehicle (UNT), ciprofloxacin (CIP), or penicillin-G (PEN) and L2-E- ftsI 3XFLAG-BMELVA infected cells were induced for FtsI at 20 hpi. Samples were fixed at 20 hpi (pre-treatment) or 30 hpi, and stained with DAPI. Images are z projected confocal micrographs showing euo prom-mNG(LVA): green, hctB prom-mKate2: red, and DAPI: cyan. A . Representative confocal micrograph of a 20 hpi. B . Representative confocal micrographs of 30 hpi UNT, CIP, PEN and FtsI-induced infections. Insert demonstrates positive anti-FLAG staining in the FtsI-induced sample. Scale bar = 10 µm. See Fig. S5 for the uninduced FtsI sample).
Figure Legend Snippet: IB to EB development is replication independent. Cos-7 cells were infected with either purified L2-BMELVA or L2-E- ftsI 3XFLAG-BMELVA. L2-BMELVA infected cells were treated with either vehicle (UNT), ciprofloxacin (CIP), or penicillin-G (PEN) and L2-E- ftsI 3XFLAG-BMELVA infected cells were induced for FtsI at 20 hpi. Samples were fixed at 20 hpi (pre-treatment) or 30 hpi, and stained with DAPI. Images are z projected confocal micrographs showing euo prom-mNG(LVA): green, hctB prom-mKate2: red, and DAPI: cyan. A . Representative confocal micrograph of a 20 hpi. B . Representative confocal micrographs of 30 hpi UNT, CIP, PEN and FtsI-induced infections. Insert demonstrates positive anti-FLAG staining in the FtsI-induced sample. Scale bar = 10 µm. See Fig. S5 for the uninduced FtsI sample).

Techniques Used: Infection, Purification, Staining

Confocal micrograph of uninduced L2-ftsI3XFLAG-BMELVA. Representative fixed confocal micrographs of Cos-7 cells infected with L2-ftsI3XFLAG-BMELVA at 20 hpi (A) and 30 hpi (B). Fixed samples were stained with DAPI and anti-FLAG. Insert demonstrates the lack of FLAG expression in both samples. Scale bar = 10 µm.
Figure Legend Snippet: Confocal micrograph of uninduced L2-ftsI3XFLAG-BMELVA. Representative fixed confocal micrographs of Cos-7 cells infected with L2-ftsI3XFLAG-BMELVA at 20 hpi (A) and 30 hpi (B). Fixed samples were stained with DAPI and anti-FLAG. Insert demonstrates the lack of FLAG expression in both samples. Scale bar = 10 µm.

Techniques Used: Infection, Staining, Expressing

FtsI overexpression induces RB cell death. Cos-7 cells were infected with L2-E-ftsI3XFLAG-BMELVA. Infected cells were induced for FtsI3XFLAG expression at 20 hpi. A. Representative confocal micrographs of a 20 hpi infection and 30 hpi uninduced and induced infections. Samples were fixed at 20 hpi (pre-treatment) or 30 hpi, stained with DAPI and an anti-FLAG antibody for immunofluorescence (IF) imaging. euoprom-mNG(LVA): green, DAPI: cyan, anti-FLAG: magenta. Scale bar = 10 µm. B. Quantification of genome copies. Uninduced (UNT): green, induced (FtsI): orange. Samples were harvested every 4 hours from 26-54 hpi. Arrow indicates time of induction. Means are shown. Cloud represents 95% ci.
Figure Legend Snippet: FtsI overexpression induces RB cell death. Cos-7 cells were infected with L2-E-ftsI3XFLAG-BMELVA. Infected cells were induced for FtsI3XFLAG expression at 20 hpi. A. Representative confocal micrographs of a 20 hpi infection and 30 hpi uninduced and induced infections. Samples were fixed at 20 hpi (pre-treatment) or 30 hpi, stained with DAPI and an anti-FLAG antibody for immunofluorescence (IF) imaging. euoprom-mNG(LVA): green, DAPI: cyan, anti-FLAG: magenta. Scale bar = 10 µm. B. Quantification of genome copies. Uninduced (UNT): green, induced (FtsI): orange. Samples were harvested every 4 hours from 26-54 hpi. Arrow indicates time of induction. Means are shown. Cloud represents 95% ci.

Techniques Used: Over Expression, Infection, Expressing, Staining, Immunofluorescence, Imaging

3) Product Images from "Membrane remodeling properties of the Parkinson’s disease protein LRRK2"

Article Title: Membrane remodeling properties of the Parkinson’s disease protein LRRK2

Journal: bioRxiv

doi: 10.1101/2022.08.10.503505

Purified LRRK2 is enzymatically active. a , Domain cartoon of the purified proteins used in the functional assays. b , Coomassie-stained SDS gels showing fractions obtained during the purification of recombinant full-length LRRK2 and RCKW fragment from Expi293 cells. Proteins were purified on anti-flag M2 resin, eluted from the beads using the FLAG peptide, followed by GST-Protease to remove the tag and further purified by removing the GST-protease by Glutathione-agarose. LRRK2- and RCKW-containing supernatants (Sup) were dialyzed before use. c , Anti-FLAG tag Western blot (WB) and Coomassie-stained gel showing successful removal of the flag tag. d , In vitro GTPase activity assay. The GTPase activity of LRRK2 (9 µM) and dynamin 1 (Dyn 1) (0.8 µM) were measured by phosphate release at saturating concentrations of GTP (0.5 mM). Left, LRRK2 has detectable GTPase activity but a much lower activity than dynamin 1. Right, Coomassie-stained SDS-PAGE showing the proteins used at the same molar ratio used for the GTPase assay. e , Protein kinase activity assay. LRRK2-mediated phosphorylation of recombinant Rab8 (upper), and autophosphorylation of LRRK2 (middle), were analyzed by Phos-tag gels using an anti-Rab8 or an anti-LRRK2 phospho-specific (pT1357) antibody, respectively. Coomassie Blue stained gels of LRRK2 samples used for the assay are shown at the bottom of the figure.
Figure Legend Snippet: Purified LRRK2 is enzymatically active. a , Domain cartoon of the purified proteins used in the functional assays. b , Coomassie-stained SDS gels showing fractions obtained during the purification of recombinant full-length LRRK2 and RCKW fragment from Expi293 cells. Proteins were purified on anti-flag M2 resin, eluted from the beads using the FLAG peptide, followed by GST-Protease to remove the tag and further purified by removing the GST-protease by Glutathione-agarose. LRRK2- and RCKW-containing supernatants (Sup) were dialyzed before use. c , Anti-FLAG tag Western blot (WB) and Coomassie-stained gel showing successful removal of the flag tag. d , In vitro GTPase activity assay. The GTPase activity of LRRK2 (9 µM) and dynamin 1 (Dyn 1) (0.8 µM) were measured by phosphate release at saturating concentrations of GTP (0.5 mM). Left, LRRK2 has detectable GTPase activity but a much lower activity than dynamin 1. Right, Coomassie-stained SDS-PAGE showing the proteins used at the same molar ratio used for the GTPase assay. e , Protein kinase activity assay. LRRK2-mediated phosphorylation of recombinant Rab8 (upper), and autophosphorylation of LRRK2 (middle), were analyzed by Phos-tag gels using an anti-Rab8 or an anti-LRRK2 phospho-specific (pT1357) antibody, respectively. Coomassie Blue stained gels of LRRK2 samples used for the assay are shown at the bottom of the figure.

Techniques Used: Purification, Functional Assay, Staining, Recombinant, FLAG-tag, Western Blot, In Vitro, Activity Assay, SDS Page, Kinase Assay

4) Product Images from "A Cell Wall Hydrolase MepH Is Negatively Regulated by Proteolysis Involving Prc and NlpI in Escherichia coli"

Article Title: A Cell Wall Hydrolase MepH Is Negatively Regulated by Proteolysis Involving Prc and NlpI in Escherichia coli

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2022.878049

MepH levels increase in the ΔmepS ΔmepM strain. (A,B) Spot dilution assay to assess the functionality of MepH-FLAG. (A) The growth phenotype of WJ126 (P ara :: mepS ΔmepM ΔnlpI mepH -FLAG) was compared with that of WJ76 (P ara :: mepS ΔmepM ΔnlpI ) and WJ95 (P ara :: mepS ΔmepM ΔnlpI ΔmepH ) by incubation on LB agar for 22 h at 30°C. (B) The growth phenotype of WJ125 (P ara :: mepS ΔmepM Δprc mepH -FLAG) was compared with that of WJ81 (P ara :: mepS ΔmepM Δprc ) and WJ89 (P ara :: mepS ΔmepM Δprc ΔmepH ) by incubation on LB agar lacking salt for 22 h at 30°C. (C) MepH-FLAG levels in the ΔmepS and ΔmepM mutants. Strains with MepH tagged with 3X FLAG at the native chromosomal locus, WJ203, WJ373 ( ΔmepS ), WJ374 ( ΔmepM ), and WJ309 ( ΔmepS ΔmepM ), were grown overnight in M9 glucose medium lacking casamino acids. The cultures were washed and diluted in LB to an OD 600 of 0.05. Cells were then grown with agitation at 30°C and harvested around an OD 600 of 0.5. Equivalent amounts of whole-cell lysates of each strain were used for immunoblotting with anti-FLAG and anti-RpoA antibodies. Shown are the representative images from triplicate experiments. (D) The band intensities of the Western blot in (C) were quantified using ImageJ software and the relative MepH-Flag levels are shown after normalization with RpoA signals. Error bars represent the standard deviation from triplicate measurements.
Figure Legend Snippet: MepH levels increase in the ΔmepS ΔmepM strain. (A,B) Spot dilution assay to assess the functionality of MepH-FLAG. (A) The growth phenotype of WJ126 (P ara :: mepS ΔmepM ΔnlpI mepH -FLAG) was compared with that of WJ76 (P ara :: mepS ΔmepM ΔnlpI ) and WJ95 (P ara :: mepS ΔmepM ΔnlpI ΔmepH ) by incubation on LB agar for 22 h at 30°C. (B) The growth phenotype of WJ125 (P ara :: mepS ΔmepM Δprc mepH -FLAG) was compared with that of WJ81 (P ara :: mepS ΔmepM Δprc ) and WJ89 (P ara :: mepS ΔmepM Δprc ΔmepH ) by incubation on LB agar lacking salt for 22 h at 30°C. (C) MepH-FLAG levels in the ΔmepS and ΔmepM mutants. Strains with MepH tagged with 3X FLAG at the native chromosomal locus, WJ203, WJ373 ( ΔmepS ), WJ374 ( ΔmepM ), and WJ309 ( ΔmepS ΔmepM ), were grown overnight in M9 glucose medium lacking casamino acids. The cultures were washed and diluted in LB to an OD 600 of 0.05. Cells were then grown with agitation at 30°C and harvested around an OD 600 of 0.5. Equivalent amounts of whole-cell lysates of each strain were used for immunoblotting with anti-FLAG and anti-RpoA antibodies. Shown are the representative images from triplicate experiments. (D) The band intensities of the Western blot in (C) were quantified using ImageJ software and the relative MepH-Flag levels are shown after normalization with RpoA signals. Error bars represent the standard deviation from triplicate measurements.

Techniques Used: Dilution Assay, Acetylene Reduction Assay, Incubation, Western Blot, Software, Standard Deviation

Prc and NlpI are involved in the negative regulation of MepH levels. (A) Comparison of DD-endopeptidase levels in the ΔmepS ΔmepM , ΔmepS ΔmepM Δprc, and ΔmepS ΔmepM ΔnlpI strains. The ΔmepS ΔmepM strains with DD-endopeptidase tagged with 3X FLAG and their Δprc and ΔnlpI derivatives were grown overnight in M9 glucose lacking casamino acids. The cells were washed and diluted in LB to an OD600 of 0.05. When the cultures reached an OD 600 of 0.5, cells were harvested by centrifugation, resuspended in Laemmli buffer, and used for immunoblotting. (B,C) In vivo degradation assay of MepH-Flag. WJ309 ( ΔmepS ΔmepM mepH-FLAG ), WJ199 ( ΔmepS ΔmepM Δprc mepH-FLAG ), and WJ200 ( ΔmepS ΔmepM ΔnlpI mepH-FLAG ) were grown to an OD 600 of 0.5 in LB, as described in (A) . Spectinomycin was added to each culture to a final concentration of 500 μg/ml to block protein synthesis and aliquots were collected at the indicated time points for immunoblotting. The anti-FLAG signal of each sample was normalized to the anti-RpoA signal. Each experiment was performed in triplicates and the representative images are shown. (C) The normalized signal at the time of spectinomycin addition was set as 1 and the change in signal intensity at each time point was plotted for each strain. Error bars represent the standard deviation from triplicate measurements.
Figure Legend Snippet: Prc and NlpI are involved in the negative regulation of MepH levels. (A) Comparison of DD-endopeptidase levels in the ΔmepS ΔmepM , ΔmepS ΔmepM Δprc, and ΔmepS ΔmepM ΔnlpI strains. The ΔmepS ΔmepM strains with DD-endopeptidase tagged with 3X FLAG and their Δprc and ΔnlpI derivatives were grown overnight in M9 glucose lacking casamino acids. The cells were washed and diluted in LB to an OD600 of 0.05. When the cultures reached an OD 600 of 0.5, cells were harvested by centrifugation, resuspended in Laemmli buffer, and used for immunoblotting. (B,C) In vivo degradation assay of MepH-Flag. WJ309 ( ΔmepS ΔmepM mepH-FLAG ), WJ199 ( ΔmepS ΔmepM Δprc mepH-FLAG ), and WJ200 ( ΔmepS ΔmepM ΔnlpI mepH-FLAG ) were grown to an OD 600 of 0.5 in LB, as described in (A) . Spectinomycin was added to each culture to a final concentration of 500 μg/ml to block protein synthesis and aliquots were collected at the indicated time points for immunoblotting. The anti-FLAG signal of each sample was normalized to the anti-RpoA signal. Each experiment was performed in triplicates and the representative images are shown. (C) The normalized signal at the time of spectinomycin addition was set as 1 and the change in signal intensity at each time point was plotted for each strain. Error bars represent the standard deviation from triplicate measurements.

Techniques Used: Centrifugation, In Vivo, Degradation Assay, Concentration Assay, Blocking Assay, Standard Deviation

5) Product Images from "Biophysical and functional characterization of the human TAS1R2 sweet taste receptor overexpressed in a HEK293S inducible cell line"

Article Title: Biophysical and functional characterization of the human TAS1R2 sweet taste receptor overexpressed in a HEK293S inducible cell line

Journal: Scientific Reports

doi: 10.1038/s41598-021-01731-3

Analysis of immunoaffinity purified hTAS1R2. FLAG-tagged hTAS1R2 was solubilized in PBS containing 2% LMNG and captured using the EZview Red anti-FLAG M2 affinity gel. After elution with FLAG peptide, the eluate was collected, concentrated and subjected to SDS-PAGE followed by ( A ) staining with Coomassie blue and ( B ) by western blotting using mouse anti-FLAG M2 primary antibody and goat anti-mouse horseradish peroxidase conjugated secondary antibody. Full-length gels/blots are presented in Supplementary Fig. S3 .
Figure Legend Snippet: Analysis of immunoaffinity purified hTAS1R2. FLAG-tagged hTAS1R2 was solubilized in PBS containing 2% LMNG and captured using the EZview Red anti-FLAG M2 affinity gel. After elution with FLAG peptide, the eluate was collected, concentrated and subjected to SDS-PAGE followed by ( A ) staining with Coomassie blue and ( B ) by western blotting using mouse anti-FLAG M2 primary antibody and goat anti-mouse horseradish peroxidase conjugated secondary antibody. Full-length gels/blots are presented in Supplementary Fig. S3 .

Techniques Used: Purification, SDS Page, Staining, Western Blot

Immunocytochemistry of hTAS1R2-inducible HEK293S cells treated with tetracycline and NaBu. Cells from clone 7 were treated with 1 µg/mL tetracycline and 5 mM NaBu for 48 h. The level of induced hTAS1R2 protein (shown in green) was detected using a primary anti-FLAG M2 antibody and a fluorescently labelled secondary antibody (Alexa Fluor 488). The cell surface (shown in red) was detected by biotin-conjugated concanavalin A and streptavidin-conjugated Alexa Fluor 568. The overlay images indicate the localization of the receptor at the cell surface (shown in yellow). ( A ) The cells were analysed using an epi-fluorescence inverted microscope (Eclipse TiE, Nikon, Champigny sur Marne, France) equipped with an ×20 objective lens and a LucaR EMCCD camera (Andor Technology, Belfast, UK). ( B ) The cells were analysed using a two photon confocal microscope (Nikon A1-MP) equipped with an ×60 objective lens (DImaCell platform, University of Burgundy, Dijon, France).
Figure Legend Snippet: Immunocytochemistry of hTAS1R2-inducible HEK293S cells treated with tetracycline and NaBu. Cells from clone 7 were treated with 1 µg/mL tetracycline and 5 mM NaBu for 48 h. The level of induced hTAS1R2 protein (shown in green) was detected using a primary anti-FLAG M2 antibody and a fluorescently labelled secondary antibody (Alexa Fluor 488). The cell surface (shown in red) was detected by biotin-conjugated concanavalin A and streptavidin-conjugated Alexa Fluor 568. The overlay images indicate the localization of the receptor at the cell surface (shown in yellow). ( A ) The cells were analysed using an epi-fluorescence inverted microscope (Eclipse TiE, Nikon, Champigny sur Marne, France) equipped with an ×20 objective lens and a LucaR EMCCD camera (Andor Technology, Belfast, UK). ( B ) The cells were analysed using a two photon confocal microscope (Nikon A1-MP) equipped with an ×60 objective lens (DImaCell platform, University of Burgundy, Dijon, France).

Techniques Used: Immunocytochemistry, Fluorescence, Inverted Microscopy, Microscopy

Size exclusion chromatography of immunoaffinity-purified hTAS1R2. ( A ) SEC analysis was performed on an Akta Pure FPLC system equipped with a Superdex 200 Increase 10/300GL column (GE Healthcare). Immunoaffinity-purified hTAS1R2 was eluted using PBS-LMNG 0.1% (pH 7.3). Six distinct peaks were observed, suggesting the presence of several oligomeric forms of the protein. The peak fractions were analysed by ( B ) SDS-PAGE followed by staining with Coomassie blue and ( C ) by western blotting using mouse anti-FLAG M2 primary antibody and goat anti-mouse horseradish peroxidase conjugated secondary antibody. The peak and fraction numbers refer to those designated in ( A ). Peak 4 (fractions 14–16) contained mainly hTAS1R2 marked with a black asterisk.
Figure Legend Snippet: Size exclusion chromatography of immunoaffinity-purified hTAS1R2. ( A ) SEC analysis was performed on an Akta Pure FPLC system equipped with a Superdex 200 Increase 10/300GL column (GE Healthcare). Immunoaffinity-purified hTAS1R2 was eluted using PBS-LMNG 0.1% (pH 7.3). Six distinct peaks were observed, suggesting the presence of several oligomeric forms of the protein. The peak fractions were analysed by ( B ) SDS-PAGE followed by staining with Coomassie blue and ( C ) by western blotting using mouse anti-FLAG M2 primary antibody and goat anti-mouse horseradish peroxidase conjugated secondary antibody. The peak and fraction numbers refer to those designated in ( A ). Peak 4 (fractions 14–16) contained mainly hTAS1R2 marked with a black asterisk.

Techniques Used: Size-exclusion Chromatography, Purification, Fast Protein Liquid Chromatography, SDS Page, Staining, Western Blot

6) Product Images from "Archaeal origins of gamete fusion"

Article Title: Archaeal origins of gamete fusion

Journal: bioRxiv

doi: 10.1101/2021.10.13.464100

Surface expression of FsxA and mutants. BHK cells were transfected with FLAG-tagged FsxA (WT) and the indicated mutants; the FLAG tag was inserted before the membrane anchor (see Extended Data Fig. 7d). Non-permeabilized staining using anti-FLAG antibody showed surface expression of FsxA and the various mutants. The proportion of non-permeabilized cells showing surface expression was: AFF-1-FLAG (negative control; 0%, n∼1000), FsxA-FLAG (3.9%, n=1242), FsxA-ΔFL→AG 4 A-FLAG (4.4%, n=1176), FsxA-ΔDIV→EFF-1stem-FLAG (2.6%, n=1263). Another group of transfected BHK cells in parallel were fixed, permeabilized and stained with anti-FLAG antibody. Permeabilized staining showed main distribution in the cytoplasm (endoplasmic reticulum) of FsxA and its mutants. C. elegans AFF-1 tagged with FLAG at the C terminus (cytoplasmic tail) worked as a negative control for non-permeabilized staining. Scale Bars, 10 µm.
Figure Legend Snippet: Surface expression of FsxA and mutants. BHK cells were transfected with FLAG-tagged FsxA (WT) and the indicated mutants; the FLAG tag was inserted before the membrane anchor (see Extended Data Fig. 7d). Non-permeabilized staining using anti-FLAG antibody showed surface expression of FsxA and the various mutants. The proportion of non-permeabilized cells showing surface expression was: AFF-1-FLAG (negative control; 0%, n∼1000), FsxA-FLAG (3.9%, n=1242), FsxA-ΔFL→AG 4 A-FLAG (4.4%, n=1176), FsxA-ΔDIV→EFF-1stem-FLAG (2.6%, n=1263). Another group of transfected BHK cells in parallel were fixed, permeabilized and stained with anti-FLAG antibody. Permeabilized staining showed main distribution in the cytoplasm (endoplasmic reticulum) of FsxA and its mutants. C. elegans AFF-1 tagged with FLAG at the C terminus (cytoplasmic tail) worked as a negative control for non-permeabilized staining. Scale Bars, 10 µm.

Techniques Used: Expressing, Transfection, FLAG-tag, Staining, Negative Control

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    Millipore monoclonal anti flag m2 antibody produced in mouse
    Only overexpression of full-length HTT can rescue HAP40 protein levels <t>in</t> HTT-null cells. A-B, Representative western blot images of total cell lysates from HEK293 wildtype and HEK293 HTT-null lines after transfection with of various <t>FLAG-tagged</t> constructs for HTT full-length (FL) and truncated species of HTT. A, Nitrocellulose membrane was incubated with rabbit <t>anti-F8A1</t> <t>antibody,</t> followed by mouse anti-tubulin to detect endogenous HAP40 and tubulin. B, Nitrocellulose membrane was incubated with mouse <t>anti-FLAG</t> antibody, followed by mouse anti-GAPDH to detect overexpressed FLAG-tagged constructs and endogenous GAPDH. Calculated molecular weight for HTT 81-1643 is 175 kDa, for HTT 2088-3144 121 kDa, and for FL HTTQ23 352 kDa. Two independent experiments were performed.
    Monoclonal Anti Flag M2 Antibody Produced In Mouse, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Only overexpression of full-length HTT can rescue HAP40 protein levels in HTT-null cells. A-B, Representative western blot images of total cell lysates from HEK293 wildtype and HEK293 HTT-null lines after transfection with of various FLAG-tagged constructs for HTT full-length (FL) and truncated species of HTT. A, Nitrocellulose membrane was incubated with rabbit anti-F8A1 antibody, followed by mouse anti-tubulin to detect endogenous HAP40 and tubulin. B, Nitrocellulose membrane was incubated with mouse anti-FLAG antibody, followed by mouse anti-GAPDH to detect overexpressed FLAG-tagged constructs and endogenous GAPDH. Calculated molecular weight for HTT 81-1643 is 175 kDa, for HTT 2088-3144 121 kDa, and for FL HTTQ23 352 kDa. Two independent experiments were performed.

    Journal: bioRxiv

    Article Title: Expanding the Huntington’s disease research toolbox; validated subdomain protein constructs for biochemical and structural investigation of huntingtin

    doi: 10.1101/2022.11.21.516512

    Figure Lengend Snippet: Only overexpression of full-length HTT can rescue HAP40 protein levels in HTT-null cells. A-B, Representative western blot images of total cell lysates from HEK293 wildtype and HEK293 HTT-null lines after transfection with of various FLAG-tagged constructs for HTT full-length (FL) and truncated species of HTT. A, Nitrocellulose membrane was incubated with rabbit anti-F8A1 antibody, followed by mouse anti-tubulin to detect endogenous HAP40 and tubulin. B, Nitrocellulose membrane was incubated with mouse anti-FLAG antibody, followed by mouse anti-GAPDH to detect overexpressed FLAG-tagged constructs and endogenous GAPDH. Calculated molecular weight for HTT 81-1643 is 175 kDa, for HTT 2088-3144 121 kDa, and for FL HTTQ23 352 kDa. Two independent experiments were performed.

    Article Snippet: Primary antibodies used include anti-HTT D7F7 (Cell Signalling Technology), anti-HAP40 54731 (Novus Biologicals) and anti-FLAG F1804 (Sigma).

    Techniques: Over Expression, Western Blot, Transfection, Construct, Incubation, Molecular Weight