Journal:

Article Title: Demonstration of the interaction of transforming growth factor beta 2 and type X collagen using a modified tandem affinity purification tag

doi: 10.1016/j.jchromb.2008.10.016

Figure Lengend Snippet: LAP, TGF-b2 latency associated peptide. F, furin-type proteinases cleavage site in TGF-β2. CBP, calmodulin-binding peptide domain of hTAP. TEV, tobacco etch virus proteinases susceptible peptide in hTAP. Flag, flag peptide epitope. BM40 the bm40 signal peptide. V5 the V5 peptide epitope. Note that the BM-40 signal peptide show in D would be released after insertion into the ER rather than by Furin cleavage.

Article Snippet: Western blot Samples from cell lysates, conditioned culture medium or protein purified by hTAP affinity chromatography were separated by SDS PAGE, electrophoretically transferred to a PVDF membrane using a semi-dry electro-transfer method and incubated with primary antibody against; the Flag tag, the V5 tag (Invitrogen, Carlsbad, CA), a rabbit polyclonal antibody against recombinant chick TGF-β2 LAP protein generated in our laboratory[ 9 ], TGF-β2 (Santa Cruz Biotechnology, Santa Cruz, CA), type X collagen (using a polyclonal antibody kindly provided by Dr. Maurizio Pacifici,[ 21 ], a monoclonal antibody against the N-terminal peptide of chick type X collagen kindly supplied by Dr. Gary Balian,[ 22 ]), or the protein A domain of the hTAP tag using the peroxidase anti peroxidase complex[ 17 ].

Techniques: Binding Assay

Journal:

Article Title: Demonstration of the interaction of transforming growth factor beta 2 and type X collagen using a modified tandem affinity purification tag

doi: 10.1016/j.jchromb.2008.10.016

Figure Lengend Snippet: A) Chick fibroblasts were transfected with retroviral constructs containing the transgene TGF-β2 fused with the original (TAP) or the humanized TAP (hTAP) tags. Conditioned culture media were collected from infected fibroblasts as well as normal fibroblasts with no infection (control), separated by SDS-PAGE, immunoblotted using an antibody to TGF-β2 and detected by chemiluminescence. The 62 kDa band present in all culture media was due to a nonspecific reaction with the primary antibody and served to indicate equal protein loading in all samples. The same amount of protein was loaded on each lane and determined by BioRad protein assay. The 33 kDa TGF-β2/TAP fusion protein was prominent in conditioned medium of fibroblasts transfected with the hTAP construct and only weakly detected in fibroblasts transfected with the original TAP construct. B) Chick chondrocytes were infected with the TGF-β2 fused with hTAP (hTAP) or TGF-β2 fused with the small Flag tag (flag). Culture medium and cell lysates were prepared, separated by SDS-PAGE and immunoblotted using the TGF-β2 antibody. Comparable protein loading was achieved by using the same volume of culture medium and cell numbers with greater than 90% transfection efficiency. Proteins detected in cell lysates had migration consistent with hTAP-tagged TGF-β2 precursor (73kDa), a slightly faster migrating protein assumed to be a breakdown product and flag-tagged TGF-β2 precursor (53 kDa). Proteins detected in the culture medium were consistent with hTAP-tagged TGF-β2 (33 kDa) and flag-tagged TGF-β2 (13 kDa)Minimal contribution of the hTAP tag to signal intensity seen in these images is expected. Mouse IgG (TGF-β2 monoclonal antibody used) has weak interaction with the protein A component of the hTAP tag.

Article Snippet: Western blot Samples from cell lysates, conditioned culture medium or protein purified by hTAP affinity chromatography were separated by SDS PAGE, electrophoretically transferred to a PVDF membrane using a semi-dry electro-transfer method and incubated with primary antibody against; the Flag tag, the V5 tag (Invitrogen, Carlsbad, CA), a rabbit polyclonal antibody against recombinant chick TGF-β2 LAP protein generated in our laboratory[ 9 ], TGF-β2 (Santa Cruz Biotechnology, Santa Cruz, CA), type X collagen (using a polyclonal antibody kindly provided by Dr. Maurizio Pacifici,[ 21 ], a monoclonal antibody against the N-terminal peptide of chick type X collagen kindly supplied by Dr. Gary Balian,[ 22 ]), or the protein A domain of the hTAP tag using the peroxidase anti peroxidase complex[ 17 ].

Techniques: Transfection, Construct, Infection, SDS Page, FLAG-tag, Migration

Journal:

Article Title: Demonstration of the interaction of transforming growth factor beta 2 and type X collagen using a modified tandem affinity purification tag

doi: 10.1016/j.jchromb.2008.10.016

Figure Lengend Snippet: A, culture medium from chondrocytes not infected with the plasmid (lane 1), DMEM, 10% FCS (lane 2), culture medium from chondrocytes infected with the RCASBP(A)cTGF-β2-hTAP construct (lane 3) was subjected to tandem affinity purification and analyzed for protein by sliver staining. Open arrows 1 and 2 indicate proteins bands analyzed by mass spectrometry. Protein bands shown by the bracket indicate contaminating keratins found in all samples. B, protein samples shown in A lane 3 were identified by Western blot using TGF-β2 LAP- (lane 1) and TGF-β2- (lane 2) specific antibodies after SDS PAGE. The migration positions of protein molecular weight standards are shown at left. C, Detection of type X collagen in complex with TGF-β2-hTAP fusion protein. Protein complexes were isolated from conditioned culture medium from control chondrocytes (lane 1) or three separate preparations of chondrocytes infected with the RCASBP TGF-β2-TAP construct (lanes 2, 3, 4) by tandem affinity purification. The presence of type X collagen was detected by Western blot after SDS PAGE using a type X collagen polyclonal antibody. Arrows to the right of the figure show predicted migration positions of type X collagen monomer, trimer and higher order multimers. The migration position of protein molecular weight standards are shown to the left.

Article Snippet: Western blot Samples from cell lysates, conditioned culture medium or protein purified by hTAP affinity chromatography were separated by SDS PAGE, electrophoretically transferred to a PVDF membrane using a semi-dry electro-transfer method and incubated with primary antibody against; the Flag tag, the V5 tag (Invitrogen, Carlsbad, CA), a rabbit polyclonal antibody against recombinant chick TGF-β2 LAP protein generated in our laboratory[ 9 ], TGF-β2 (Santa Cruz Biotechnology, Santa Cruz, CA), type X collagen (using a polyclonal antibody kindly provided by Dr. Maurizio Pacifici,[ 21 ], a monoclonal antibody against the N-terminal peptide of chick type X collagen kindly supplied by Dr. Gary Balian,[ 22 ]), or the protein A domain of the hTAP tag using the peroxidase anti peroxidase complex[ 17 ].

Techniques: Infection, Plasmid Preparation, Construct, Affinity Purification, Staining, Mass Spectrometry, Western Blot, SDS Page, Migration, Molecular Weight, Isolation

Journal:

Article Title: Demonstration of the interaction of transforming growth factor beta 2 and type X collagen using a modified tandem affinity purification tag

doi: 10.1016/j.jchromb.2008.10.016

Figure Lengend Snippet: Interaction of type X collagen with TGF-β2 was monitored by Western blot using LAP-specific (A) and V5-specific (B) antibodies after isolation by calmodulin affinity chromatography. Lane 1 to lane 4 conditioned culture medium from 293 cells expressing both type X collagen fused with the hTAP tag and V5 tagged TGF-β2. Lane 5 conditioned culture medium from 293 cells expressing V5 tagged TGF-β2 only. The IgG binding domain of the type X collagen hTAP fusion protein was detected by the second antibody used to detect the LAP-specific rabbit polyclonal antibody in A. The bands migrating at 50–60 kDa in Bare calmodulin binding proteins present in the bovine serum component of culture medium that react with the second antibody. Neither TGF-β2 LAP (A) nor TGF-β2 (B) bound to the calmodulin column in the absence of type X collagen (lane 2). Controls shown in lane 5 were separated on the same gel but lane sequences have been shifted to improve readability.

Article Snippet: Western blot Samples from cell lysates, conditioned culture medium or protein purified by hTAP affinity chromatography were separated by SDS PAGE, electrophoretically transferred to a PVDF membrane using a semi-dry electro-transfer method and incubated with primary antibody against; the Flag tag, the V5 tag (Invitrogen, Carlsbad, CA), a rabbit polyclonal antibody against recombinant chick TGF-β2 LAP protein generated in our laboratory[ 9 ], TGF-β2 (Santa Cruz Biotechnology, Santa Cruz, CA), type X collagen (using a polyclonal antibody kindly provided by Dr. Maurizio Pacifici,[ 21 ], a monoclonal antibody against the N-terminal peptide of chick type X collagen kindly supplied by Dr. Gary Balian,[ 22 ]), or the protein A domain of the hTAP tag using the peroxidase anti peroxidase complex[ 17 ].

Techniques: Western Blot, Isolation, Affinity Chromatography, Expressing, Binding Assay

Journal:

Article Title: Demonstration of the interaction of transforming growth factor beta 2 and type X collagen using a modified tandem affinity purification tag

doi: 10.1016/j.jchromb.2008.10.016

Figure Lengend Snippet: EBNA 293 were co-transfected with hTAP-tagged type X collagen and V5-tagged TGF-β2. Concentrations of type X collagen DNA transfected as a ratio of TGF-β2 transfected ranged from 0 to 8 as indicated, while maintaining a constant level of TGF-β2 DNA. TGF-beta activity was measured in culture medium by mink lung cell bioassay, after heat activation. Average and standard deviation of TGF-β activity expressed as a percentage of control from 4 cultures is shown.

Article Snippet: Western blot Samples from cell lysates, conditioned culture medium or protein purified by hTAP affinity chromatography were separated by SDS PAGE, electrophoretically transferred to a PVDF membrane using a semi-dry electro-transfer method and incubated with primary antibody against; the Flag tag, the V5 tag (Invitrogen, Carlsbad, CA), a rabbit polyclonal antibody against recombinant chick TGF-β2 LAP protein generated in our laboratory[ 9 ], TGF-β2 (Santa Cruz Biotechnology, Santa Cruz, CA), type X collagen (using a polyclonal antibody kindly provided by Dr. Maurizio Pacifici,[ 21 ], a monoclonal antibody against the N-terminal peptide of chick type X collagen kindly supplied by Dr. Gary Balian,[ 22 ]), or the protein A domain of the hTAP tag using the peroxidase anti peroxidase complex[ 17 ].

Techniques: Transfection, Activity Assay, Activation Assay, Standard Deviation

Journal:

Article Title: Demonstration of the interaction of transforming growth factor beta 2 and type X collagen using a modified tandem affinity purification tag

doi: 10.1016/j.jchromb.2008.10.016

Figure Lengend Snippet: Paraffin sections of iliac crest growth cartilage were incubated with an antibody against TGF-β2 and the vector red substrate. A low power view, B high power view top normal growth cartilage, bottom SMCD growth cartilage. TGF-β2 is localized in close association with hypertrophic chondrocytes adjacent to the resorbing vasculature in the unaffected child in contrast to a pericellular and faint diffuse inter-territorial distribution in the in the SMCD child.

Article Snippet: Western blot Samples from cell lysates, conditioned culture medium or protein purified by hTAP affinity chromatography were separated by SDS PAGE, electrophoretically transferred to a PVDF membrane using a semi-dry electro-transfer method and incubated with primary antibody against; the Flag tag, the V5 tag (Invitrogen, Carlsbad, CA), a rabbit polyclonal antibody against recombinant chick TGF-β2 LAP protein generated in our laboratory[ 9 ], TGF-β2 (Santa Cruz Biotechnology, Santa Cruz, CA), type X collagen (using a polyclonal antibody kindly provided by Dr. Maurizio Pacifici,[ 21 ], a monoclonal antibody against the N-terminal peptide of chick type X collagen kindly supplied by Dr. Gary Balian,[ 22 ]), or the protein A domain of the hTAP tag using the peroxidase anti peroxidase complex[ 17 ].

Techniques: Incubation, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins *

doi: 10.1074/jbc.M116.730804

Figure Lengend Snippet: RegIIA inhibition of α3-containing nAChR subtypes and subtype mutants IC 50 values (n m ) with 95% CI. Hill slope ( n H ) was obtained from concentration–response curves for RegIIA at wild-type and mutant human α3β2 and α3β4 nAChR subtypes. Human α3β2 nAChR mutations hα3β2[T59K] and hα3β2[S113R] notably decrease the IC 50 of RegIIA towards the human α3β4 subtype value, whereas the opposite human α3β4[K59T] and α3β4[R113S] result in lower potency of RegIIA and significantly increased IC 50 values. Data from wild-type nAChRs and the mutants mentioned are highlighted in bold font. All data represent mean of n = 3–9 experiments.

Article Snippet: Site-directed Mutagenesis Plasmid DNAs encoding human and rat α3, β2, and β4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously ( 17 ).

Techniques: Inhibition, Concentration Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins *

doi: 10.1074/jbc.M116.730804

Figure Lengend Snippet: Sequence and structural comparison between human β2 and β4 nAChR ECDs. A, amino acid sequence alignment of the N-terminal ECDs of human β2 and β4 nAChR subunits shows 70% identity between the sequences (149 of the 213 residues homologous) and reveals several non-conserved residues in the ACh binding loops. Conserved residues are indicated with dots. Residues that were mutated to the opposite β subunit residue in this study are framed with red lines. Longer sequences that were replaced in hβ2 with the sequences of hβ4 are framed with green lines. Blue bars indicate ACh binding domain loops D, E, and F of the complementary interface (34). B, overlay of the hα3β2 and hα3β4 inter-subunit interfaces to emphasize the overall high structural similarity between them. The α3(+) interface is shown in green, β2(−) in pink, and β4(−) in cyan. Non-conserved residues from the complementary β2(−) and β4(−) subunits, respectively, are shown as licorice models and labeled.

Article Snippet: Site-directed Mutagenesis Plasmid DNAs encoding human and rat α3, β2, and β4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously ( 17 ).

Techniques: Sequencing, Comparison, Binding Assay, Residue, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins *

doi: 10.1074/jbc.M116.730804

Figure Lengend Snippet: The non-homologous residues at position 59 (Lys in hβ4, Thr in hβ2) and 113 (Arg in hβ4, Ser in hβ2) of the human β subunits are the key residues determining the selectivity profile in inhibitory potency of α-conotoxin RegIIA. A and B, concentration-response curves for RegIIA inhibition of wild-type and mutant human α3β2 (A) and α3β4 nAChRs (B). The two hα3β2 mutants exhibited a shift of the curve to the left compared with wild-type hα3β2 nAChR, indicating an increase in affinity to the peptide, whereas the opposite mutants of hα3β4 exhibited a lower affinity to RegIIA compared with wild-type hα3β4 nAChR. C, representative superimposed ACh-evoked currents obtained in the absence (control, black line) and presence of 100 nm RegIIA at wild-type hα3β2 (red), loop D mutant hα3β2[T59K] (green), and loop E mutant hα3β2[S113R] (blue). D, representative superimposed ACh-evoked currents obtained in the absence (control, black line) and presence of 100 nm RegIIA at wild-type (red) and mutant hα3β4 nAChRs (green and blue, respectively. E, bar graph summarizing the IC50 values with 95% CI obtained from concentration-response curves for RegIIA at wild-type hα3β2, hα3β4, and the mutants and chimeric subtypes analyzed. A gain in sensitivity was observed at mutants hα3β2[T59K] and hα3β2[S113R], whereas the most prominent reductions in sensitivity can be mapped to the opposite mutations hα3β4[K59T] and hα3β4[R113S]. Dotted squares indicate the agonist binding loops in which the respective mutants are located. All data points represent mean ± 95% CI. The IC50, 95% CI, and Hill slope (nH) values are summarized in Table 1.

Article Snippet: Site-directed Mutagenesis Plasmid DNAs encoding human and rat α3, β2, and β4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously ( 17 ).

Techniques: Concentration Assay, Inhibition, Mutagenesis, Control, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins *

doi: 10.1074/jbc.M116.730804

Figure Lengend Snippet: ACh EC 50 values for wild-type and mutant human α3β2 and α3β4 nAChRs EC 50 values (μ m ) with 95% CI. The Hill slope ( n H ) was obtained from concentration–response curves for ACh at wild-type and mutant human α3β2 and α3β4 nAChR subtypes. ACh concentrations from 0.01 μ m to 10 m m (the highest applicable concentration) were tested. All data represent mean of n = 4–8 experiments.

Article Snippet: Site-directed Mutagenesis Plasmid DNAs encoding human and rat α3, β2, and β4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously ( 17 ).

Techniques: Mutagenesis, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins *

doi: 10.1074/jbc.M116.730804

Figure Lengend Snippet: Wild-type human α3β2 and α3β4 nAChRs display different wash-off kinetics from block by α-conotoxin RegIIA. Single residue β2 mutations in hα3β2 are sufficient to switch off-rates to those of the opposite subtype. A, graph summarizing the wash-off kinetics data. The hα3β2 nAChR subtype (black circles) exhibits fast recovery of ACh-evoked currents from block by RegIIA, with full recovery achieved in less than 2 min. In contrast, at hα3β4 (black squares) currents recovered from RegIIA block after a 13–14-min washout. When loop D residue 59 in hβ2 was replaced with the respective residue of hβ4 (mutant hα3β2[T59K], green circles) the off-rate of RegIIA was dramatically slowed. A similar replacement of loop E residue 113 in hβ2 (mutant hα3β2[S113R], blue circles) slowed the recovery rate similar to hα3β4. Representative ACh-evoked currents of hα3β2 (B), hα3β2[T59K] (C), and hα3β2[S113R] (D) illustrate the recovery from the RegIIA block differs between wild-type and the two mutant hα3β2 nAChR subtypes. Numbers at the respective ACh-evoked current peaks indicate the duration (in min) of washout and C indicates a representative control ACh application before incubation with the peptide. Oocytes were incubated with RegIIA for 5 min followed by repetitive application of ACh under continuous perfusion with ND96 solution. Approximate EC50 values for ACh and RegIIA concentrations giving major to full block of ACh-evoked currents under these conditions were used for each subtype tested. All data points in A represent mean ± 95% CI, n = 3–7. The times required to reach 95% recovery from block are summarized in Table 3.

Article Snippet: Site-directed Mutagenesis Plasmid DNAs encoding human and rat α3, β2, and β4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously ( 17 ).

Techniques: Blocking Assay, Residue, Mutagenesis, Control, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins *

doi: 10.1074/jbc.M116.730804

Figure Lengend Snippet: Single residue β4 mutations in hα3β4 increase the α-conotoxin off-rate of RegIIA to values similar to wild-type hα3β2. A, graph summarizing the wash-off kinetics data. Wild-type hα3β2 and hα3β4 data are the same as described in the legend to Fig. 3 and shown for comparison. Replacing loop D residue 59 in hβ4 with the respective residue in hβ2 (mutant hα3β4[K59T], green squares) is sufficient to shift the off-rate to the opposite subtype. A mutant in which residue Arg113 of hβ4 is replaced with Ser, as in hβ2, (hα3β4[R113S], blue squares) similarly exhibits a fast wash-off rate resembling hα3β2. Representative ACh-evoked currents of hα3β4 (B), hα3β4[K59T] (C), and hα3β4[R113S] (D) illustrate the recovery from the RegIIA block differs between wild-type and the two mutant hα3β4 nAChR subtypes. Experimental conditions were the same as those described in the legend to Fig. 3. All data points in A represent mean ± 95% CI, n = 3–7. The times required to reach 95% recovery from block are summarized in Table 3.

Article Snippet: Site-directed Mutagenesis Plasmid DNAs encoding human and rat α3, β2, and β4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously ( 17 ).

Techniques: Residue, Comparison, Mutagenesis, Blocking Assay

Journal: The Journal of Biological Chemistry

Article Title: Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins *

doi: 10.1074/jbc.M116.730804

Figure Lengend Snippet: Contacts of α-conotoxin RegIIA with hα3β2 and hα3β4 nAChR, respectively Contacts between nAChR and RegIIA are defined as van der Waals interactions if the distance between heavy atoms of RegIIA and nAChR is between 2.6 and 4 Å. Residues of the nAChR forming hydrogen bonds with RegIIA are underlined. Residues that are non-conserved between α3β2 and α3β4 nAChRs, as well as RegIIA residues making contact with them are shown in bold.

Article Snippet: Site-directed Mutagenesis Plasmid DNAs encoding human and rat α3, β2, and β4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously ( 17 ).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins *

doi: 10.1074/jbc.M116.730804

Figure Lengend Snippet: α-Conotoxin AuIB has minor activity at human α3β2 and α3β4 nAChRs. A, bar graph representing potency of block by α-conotoxin AuIB (1 μm) at wild-type and mutant human α3β2 and α3β4 nAChRs. Data represent mean ± S.E., n = 3–9. B, concentration-response analysis of AuIB at wild-type α3β2 and α3β4 nAChRs indicated the IC50 is considerably higher than 30 μm at both nAChR subtypes. AuIB (30 μm) reduced ACh-evoked current amplitude mediated by α3β2 to 75.9 ± 3.7% of control (n = 10) and α3β4 currents to 79.6 ± 4.0% (n = 10), respectively. C and D, molecular dynamics simulation predicted binding modes of AuIB to α3β2 (C) and α3β4 (D). Several hydrogen bonds are formed between pairwise interacting residues of different loops and between toxin and receptor, e.g. AuIB Asp14 with β4 Arg113 (dashed circle, hydrogen bonds as dotted lines). The α3(+) interface is shown in green, β2(−) in pink, β4(−) in cyan, and AuIB in orange. Non-conserved residues are shown as licorice models and labeled. Residues from the receptor and AuIB are labeled using normal and italic fonts, respectively.

Article Snippet: Site-directed Mutagenesis Plasmid DNAs encoding human and rat α3, β2, and β4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously ( 17 ).

Techniques: Activity Assay, Blocking Assay, Mutagenesis, Concentration Assay, Control, Binding Assay, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins *

doi: 10.1074/jbc.M116.730804

Figure Lengend Snippet: Contacts of α-conotoxin AuIB with hα3β2 and hα3β4 nAChRs, respectively Contacts between nAChR and AuIB are defined as van der Waals interactions if the distance between heavy atoms of AuIB and nAChR is between 2.6 and 4 Å. Residues of the nAChR forming hydrogen bonds with AuIB are underlined. Residues that are non-conserved between α3β2 and α3β4 nAChRs, as well as AuIB residues making contact with them are shown in bold.

Article Snippet: Site-directed Mutagenesis Plasmid DNAs encoding human and rat α3, β2, and β4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously ( 17 ).

Techniques:

Journal: American Journal of Cancer Research

Article Title: LPPR4 promotes peritoneal metastasis via Sp1/integrin α/FAK signaling in gastric cancer

doi:

Figure Lengend Snippet: LPPR4 activates transcription of integrin α through Sp1. A. Classical members of integrin family proteins and corresponding downstream genes were assessed by Western blotting in HGC27 and MKN74 cells transfected with siLPPR4 and in MGC803 cells infected with LPPR4 overexpression plasmid. B. Immunoprecipitation using a LPPR4 antibody showed LPPR4 and ITGA1, ITGA2, ITGA5, ITGA6, ITGA7 association. C. Correlation between LPPR4 and Sp1, as well as Sp1 and ITGA1, ITGA2, ITGA5, ITGA6, ITGA7 was performed in human GC tissues based on TCGA-STAD dataset. D. Sp1 mRNA expression levels were tested by qRT-PCR in HGC-27 and MKN74 cells after transfected with siLPPR4. E. Sp1 protein expression levels were detected by Western blotting analysis in HGC27 and MKN74 cells after transfected with siLPPR4. F, G. HGC27 and MKN74 cells were transfected with NC-siRNA and Sp1-siRNA. Sp1 expression levels were detected by qRT-PCR and Western blotting. ITGA1, ITGA2, ITGA5, ITGA6 and ITGA7 protein expression levels were detected by Western blotting analysis in HGC27 and MKN74 cells after transfected with siSp1. β-actin was used as a loading control in Western blotting. Each experiment was repeated at least three times. All the data were expressed as mean ± SD, ***P < 0.001, based on Student’s t-test.

Article Snippet: Rabbit anti-Akt (#9272S), anti-phospho-Akt (Ser473) (#9271L), anti-Src (#2110S), anti-phospho-Src (#6943S), anti-FAK (#3285S), anti-phospho-FAK (Y397) (#3281S), anti-integrin β1 (#9699S), anti-integrin β2 (#73663S), anti-integrin β3 (#13166S), anti-integrin α5 (98204S), anti-Sp1 (#9389S) and mouse anti-β-actin (#3700S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Transfection, Infection, Over Expression, Plasmid Preparation, Immunoprecipitation, Expressing, Quantitative RT-PCR

Journal: Journal of molecular cell biology

Article Title: METTL3-mediated m6A modification of HMGA2 mRNA promotes subretinal fibrosis and epithelial-mesenchymal transition.

doi: 10.1093/jmcb/mjad005

Figure Lengend Snippet: Figure 3 METTL3 is involved in primary mouse RPE cells undergoing EMT. ( A and B ) The mRNA ( A ) and protein ( B ) levels of EMT-related markers ( E-cadherin, N-cadherin, and α-SMA ) and METTL3 in primary mouse RPE cells with and without 10 ng/ml TGF- β2 for 48 h. ( C ) Phase- contrast microscopy images of RPE cells showed the spindle fibroblast-like morphology induced by TGF- β2 application. Scale bar, 50 μm. ( D ) Immunofluorescence analysis demonstrated increased METTL3 in RPE cells treated with TGF- β2. Scale bar, 25 μm. ( E ) Primary RPE

Article Snippet: For the EMT group, cells were passaged at a 1:3 ratio and treated with recombinant mouse TGF- β2 ( R&D Systems ) at a concentration of 10 ng/ml to model the EMT of RPE in vivo .

Techniques: Microscopy

Journal: Journal of molecular cell biology

Article Title: METTL3-mediated m6A modification of HMGA2 mRNA promotes subretinal fibrosis and epithelial-mesenchymal transition.

doi: 10.1093/jmcb/mjad005

Figure Lengend Snippet: Figure 4 METTL3-mediated m 6 A modification enhances the stability of HMGA2 mRNA. ( A ) MeRIP–qRT-PCR with anti-m 6 A antibody was performed to determine the m 6 A enrichment of HMGA2 mRNA with or without METTL3 knockdown in primary RPE cells. ( B ) Western blotting analysis showed that HMGA2 protein level was induced by TGF- β2 and inhibited upon METTL3 knockdown. ( C ) Immunofluorescence analysis confirmed the reduced expression of HMGA2 in METTL3-knockdown RPE cells ( n = 15 for each group ) . Actin cytoskeleton was visualized by phalloidin staining, which targets F-actin. Scale bar, 25 μm. ( D ) Schematic representation of pmirGLO dual-luciferase vectors fused with WT or MUT HMGA2 3 ′ UTR. ( E ) 3 ′ UTR WT or 3 ′ UTR MUT reporters were transfected into RPE cells along with empty vectors or METTL3 expression plasmid. Relative luciferase activity was measured after 48 h. ( F ) After transfection with either si-METTL3 or si-NC for 48 h, primary RPE cells were treated with Act-D to block transcription. HMGA2 mRNA was analyzed at the indicated times. Data present mean ± SD of three independent experiments. Student’s t-test for two independent groups, one-way ANOVA tests for luciferase reporter assay and repeated measures, two-way ANOVA tests for HMGA2 mRNA decay experiment, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For the EMT group, cells were passaged at a 1:3 ratio and treated with recombinant mouse TGF- β2 ( R&D Systems ) at a concentration of 10 ng/ml to model the EMT of RPE in vivo .

Techniques: Quantitative RT-PCR, Knockdown, Western Blot, Expressing, Staining, Luciferase, Transfection, Plasmid Preparation, Activity Assay, Blocking Assay, Reporter Assay

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet: ( A ) HEK293T cells were transfected with nAChR subunits (α4 ( CHRNA4 ) and β2 ( CHRNB2 )) and empty vector (EV) control or SERPINH1 cDNA plasmids. Forty-eight hours post transfection, cells were lysed and the total proteins were evaluated using a western blot analysis. ( B ) Hsp47 overexpression increases FRET efficiency between CFP-tagged β2 subunit and YFP-tagged α4 subunit of nAChRs. HEK293T cells were transfected with CFP-tagged β2 subunit and YFP-tagged α4 subunit; in addition, cells were transfected with empty vector (EV) control or SERPINH1 cDNA. Forty-eight hours post transfection, pixel-based FRET was used to measure the FRET efficiency between β2-CFP and α4-YFP by using a confocal microscope. Representative images were shown for the CFP channel (1st columns), YFP channel (2nd columns), and FRET efficiency (3rd columns). Scale bar = 10 μm. Quantification of the FRET efficiency from 60 to 70 cells from at least three transfections was achieved using the ImageJ PixFRET plug-in, and shown on the right. ( C ) HEK293T cells were transfected with CFP-tagged β2 subunit and YFP-tagged α4 subunit of nAChRs; in addition, cells were transfected with empty vector (EV) control or SERPINH1 cDNA. Forty-eight hours post transfection, whole-cell patch clamping was performed to record nicotine-induced currents. Representative traces were shown. Quantification of the peak currents ( I max ) from 9 cells from three transfections is shown on the right. The holding potential was set at –60 mV. pA: picoampere. ( D ) HEK293T cells were transfected with 5-HT 3 R subunits (5-HT 3 A and FLAG-tagged 5-HT 3 B) and empty vector (EV) control or SERPINH1 cDNA plasmids. Forty-eight hours post transfection, the surface proteins were measured using a cell surface protein biotinylation assay, and the total proteins were evaluated using a Western blot analysis. The Na + /K + ATPase serves as a loading control for biotinylated membrane proteins. Three biological replicates were performed. Each data point is reported as mean ± SD. Statistical significance was calculated using two-tailed Student’s t-Test. * p<0.05; ** p<0.01; *** p<0.001. Figure 7—source data 1. Original files for the western blot analysis in . Figure 7—source data 2. PDF containing the original blots in with the relevant bands clearly labeled. Figure 7—source data 3. Data used for graphs presented in .

Article Snippet: The nAChR α4 ( CHRNA4 ) plasmid (Addgene plasmid # 24271; http://n2t.net/addgene :24271; RRID: Addgene_24271 ) and nAChR β2 ( CHRNB2 ) plasmid were a gift from Henry Lester (Addgene plasmid # 24272; http://n2t.net/addgene :24272; RRID: Addgene_24272 ).

Techniques: Transfection, Plasmid Preparation, Control, Western Blot, Over Expression, Microscopy, Cell Surface Biotinylation Assay, Membrane, Two Tailed Test, Labeling

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet:

Article Snippet: The nAChR α4 ( CHRNA4 ) plasmid (Addgene plasmid # 24271; http://n2t.net/addgene :24271; RRID: Addgene_24271 ) and nAChR β2 ( CHRNB2 ) plasmid were a gift from Henry Lester (Addgene plasmid # 24272; http://n2t.net/addgene :24272; RRID: Addgene_24272 ).

Techniques: Transfection, Construct, Control, Recombinant, Plasmid Preparation, Cloning, Software

Journal: Molecular Cancer

Article Title: Catecholamine up-regulates MMP-7 expression by activating AP-1 and STAT3 in gastric cancer

doi: 10.1186/1476-4598-9-269

Figure Lengend Snippet: Isoproterenol up-regulates MMP-7 promoter activity and activates STAT3 and AP-1 . A, schematic representation of the plasmid pMMP-7 containing a luciferase reporter gene driven by a 340 bp MMP-7 promoter fragment. SBE, STAT3 site; ABE, AP-1 site. B, MGC-803 cells were co-transfected with pMMP-7 and pRL-TK. After transfection for 48 h, the cells were incubated in serum-free medium for an additional 24 h and then stimulated with 10 μM isoproterenol for 0, 1, 2, 3, 6 or 9 h, respectively. MMP-7 promoter activities were assessed by luciferase assays. C and D, MGC-803 cells were treated with 10 μM propranolol for 1 h and then with 10 μM isoproterenol for 0, 15, 30, 60, 120 or 180 min, respectively. The phosphorylation of STAT3 and c-Jun was analyzed by Western blot with anti-phosphor-STAT3 and anti-phosphor-c-Jun rabbit polyclonal antibodies. ISO, isoproterenol.

Article Snippet: The antibodies used include anti-β2-AR rabbit polyclonal antibody (Santa Cruz Biotechnology Inc.), anti-MMP-7 mouse monoclonal antibody (Santa Cruz Biotechnology Inc.), anti-STAT3 rabbit polyclonal antibody (Cell Signaling Technology Inc.), anti-phosphor-STAT3 (Tyr705) rabbit polyclonal antibody (Cell Signaling Technology Inc.), anti-phosphor-c-Jun (Ser73) rabbit polyclonal antibody (Cell Signaling Technology Inc.), anti-β2-AR rabbit polyclonal antibody (Santa Cruz Biotechnology Inc.), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rabbit monoclonal antibody (Cell Signaling Technology Inc.).

Techniques: Activity Assay, Plasmid Preparation, Luciferase, Transfection, Incubation, Phospho-proteomics, Western Blot

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation

doi: 10.3390/cells11091532

Figure Lengend Snippet: Integrin β2 can be substituted by its human homolog in mouse Hoxb8 FL cells. ( A ) Scheme of workflow. Hoxb8 FL cells were generated from bone marrow of integrin β2-deficient mice and retrovirally transduced with mouse or human integrin β2. Human/mouse integrin β2-positive cells were FACS-sorted. ( B ) Surface expression of different integrin subunits and cell markers on neutrophil-like cells differentiated from control (β2 +/+ ), integrin β2 ko (β2 −/− ), and human or mouse integrin β2-rescued integrin β2 −/− (β2 −/− /hβ2 and β2 −/− /mβ2) Hoxb8 FL cells assessed by FACS analysis. ( C ) Integrin β2 surface expression levels on neutrophil-like cells differentiated from integrin β2 −/− Hoxb8 FL cells retrovirally transduced with human integrin β2 (β2 −/− /hβ2) compared to PMNs isolated from human blood. ( D ) Static adhesion of untreated, TNFα-treated, or PMA-treated Hoxb8 FL-derived β2 +/+ , β2 −/− , β2 −/− /hβ2, and β2 −/− /mβ2 neutrophils on ICAM1. N = 4. Individual data points of the 4 independent experiments are shown. ( E , F ) Adhesion ( E ) and rolling velocities ( F ) of neutrophil-like cells differentiated from β2 +/+ , β2 −/− , β2 −/− /hβ2, and β2 −/− /mβ2 Hoxb8 FL cells in flow chambers coated with ICAM1 and P-selectin with or without CXCL1 under constant shear rate of 1 dyn/cm 2 . N = 10/11 chambers with CXCL1 (rolling and adhesion); N = 3/4 without CXCL1 (adhesion). ( G ) Neutrophil-like β2 +/+ , β2 −/− , β2 −/− /hβ2, and β2 −/− /mβ2 cells plated on a P-selectin-, ICAM1-, and CXCL1-coated surface for 10 min. Scale bar: 100 µm. All values are given as mean ± SD. * p < 0.05, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Generated, Transduction, Expressing, Control, Isolation, Derivative Assay, Shear

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation

doi: 10.3390/cells11091532

Figure Lengend Snippet: Expression of human integrin β2 rescues spreading and adhesion defects in mouse integrin β2 knockout macrophages. ( A ) Adhesion of integrin β2 −/− and human or mouse integrin β2 expressing integrin β2 −/− macrophages to ICAM1 and fibronectin in relation to control macrophages. N = 5. Individual data points represent the 5 independent experiments. ( B ) Spreading of control, integrin β2 −/− , and human or mouse integrin β2 expressing integrin β2 −/− macrophages to ICAM1 and fibronectin assessed 2 h after plating. N = 3 independent experiments, shown as individual data points. ( C ) Hoxb8-derived β2 +/+ , β2 −/− , β2 −/− /hβ2, and β2 −/− /mβ2 macrophages plated on an ICAM1- or fibronectin-coated surface for 2 h. Scale bar: 100 µm. ( D ) Alignment of the amino acid sequence of the mouse and human β2 integrin cytoplasmic tails. Talin-binding NPLF and kindlin-binding NPKF motives are highlighted in red. MP, membrane proximal; MD, membrane distal. ( E ) Control, integrin β2 −/− , and human or mouse integrin β2 expressing integrin β2 −/− macrophages were either kept in suspension or plated on ICAM1 for 20 min to assess integrin-mediated signaling. Western blot analyses for Y402-phosphorylated and total Pyk2; Y31-phosphorylated and total paxillin; T202/Y204-phosphorylated and total ERK1/2; talin; kindlin3; and general tyrosine phosphorylation. GAPDH served as loading control. All values are given as mean ± SD. *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Expressing, Knock-Out, Control, Derivative Assay, Sequencing, Binding Assay, Membrane, Suspension, Western Blot, Phospho-proteomics

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation

doi: 10.3390/cells11091532

Figure Lengend Snippet: Expression of human integrin β2 in mouse integrin β2 −/− Hoxb8 cells allows assessment of integrin activity using conformation-specific anti-human integrin β2 antibodies. ( A , B ) FACS analyses of integrin β2 activation of Hoxb8-derived neutrophils expressing human or mouse integrin β2 by measuring staining intensities of conformation-specific antibodies mAb24 ( A ) and KIM127 ( B ) either untreated, EDTA-treated, or in response to TNFα, fMLP, or PMA. ( C , D ) Relative mAb24 ( C ) and KIM127 ( D ) binding to resting or EDTA-, TNFα-, fMLP-, or PMA-stimulated Hoxb8-derived neutrophil-like cells upon treatment with DMSO, the phosphatidylinositol-3-kinase (PI3K) inhibitor Wortmannin, the phospholipase C (PLC) inhibitor U-73122, or the protein kinase C (PKC) inhibitor Gö6983. N = 7 (mAb24) or 5 (KIM127) independent experiments, shown as individual data points. All values are given as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Expressing, Activity Assay, Activation Assay, Derivative Assay, Staining, Binding Assay

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation

doi: 10.3390/cells11091532

Figure Lengend Snippet: Both talin1 and kindlin3 are required for β2 integrin activation. ( A ) Scheme of workflow. Talin1, kindlin3, and talin1/kindlin3 double-deficient Hoxb8 FL cells were generated via the CRISPR/Cas9 system. Single cell clones were screened for talin1 and kindlin3 expression. Four clones per genotype were subjected to CRISPR/Cas9-mediated integrin β2 ablation and retrovirally transduced to express human integrin β2. Mouse integrin β2-negative and human integrin β2-positive cells were FACS-sorted. ( B ) Western blot analysis of neutrophil-like cells differentiated from different human integrin β2 expressing Hoxb8 single cell clones, in which talin1 and/or kindlin3 were ablated with the CRISPR/Cas9 system. GAPDH served as loading control. ( C , D ) Relative mAb24 ( C ) and KIM127 ( D ) binding to neutrophil-like cells derived from Hoxb8 single cell clones, expressing human integrin β2 and lacking talin1 and/or kindlin3 expression. N = 4 clones. Result of each clone is plotted as individual data point. All values are given as mean ± SD. ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Activation Assay, Generated, CRISPR, Clone Assay, Expressing, Western Blot, Control, Binding Assay, Derivative Assay

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation

doi: 10.3390/cells11091532

Figure Lengend Snippet: Kindlin3 is dispensable for P-selectin-induced integrin αLβ2-mediated slow rolling but required for chemokine-induced slower rolling. ( A – C ) Adhesion ( A ), rolling velocities ( B ), and rolling velocities after LFA-1 blocking ( C ) of Hoxb8 cell-derived PMN-LCs in flow chambers coated with ICAM1, P-selectin, and CXCL1 under constant shear rate of 1 dyn/cm 2 . N = 12–16 ( A , B ) and 6–9 ( C ) flow chambers. ( D ) Rolling velocities of PMN-LCs on ICAM1- and P-selectin-coated surfaces (without CXCL1) under constant shear rate of 1 dyn/cm 2 . N = 8–9 flow chambers. Cells were differentiated from different single cell clones, in which talin1 and/or kindlin3 were ablated with the CRISPR/Cas9 system. Rolling velocities are shown as cumulative distribution of the velocities of approximately 500 ( B ), 300 ( C ), and 400 ( D ) cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Blocking Assay, Derivative Assay, Shear, Clone Assay, CRISPR

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation

doi: 10.3390/cells11091532

Figure Lengend Snippet: Talin and/or kindlin3 binding-deficient human integrin β2 exhibit impaired integrin activation. ( A ) Scheme of the integrin β2 cytoplasmic domain, which binds talin1 via a membrane proximal NPLF motif and kindlin3 via a membrane-distal NPKF and a TTT motif ( left ). Mutation of these motifs to NPLA and AAA prevent talin1 and kindlin3 binding, respectively ( right ). ( B ) Relative mAb24 binding to neutrophil-like cells derived from Hoxb8 cells expressing human wild-type integrin β2, mutant integrin β2 F/A (F754A), mutant integrin β2 TTT/AAA (T758A, T759A, T760A), or double-mutant integrin F/A TTT/AAA in response to TNFα, CXCL1, fMLP, and PMA or left untreated. N = 5 experiments indicated as individual data points. All values are given as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Binding Assay, Activation Assay, Membrane, Mutagenesis, Derivative Assay, Expressing

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation

doi: 10.3390/cells11091532

Figure Lengend Snippet: Rapid CRISPR-based screening approach to study putative β2 integrin regulators. ( A ) Scheme of workflow. Integrin β2 −/− Hoxb8 cells expressing human integrin β2 were electroporated with a control Cas9/gRNA complex (non-targeting control, NTC) or a Cas9/gRNA complex targeting a gene of interest. Cells were analyzed for integrin activity in bulk after differentiation into neutrophil-like cells. ( B ) Western blot analysis of talin1 and kindlin3 expression in Hoxb8-derived neutrophil-like cells transfected with Cas9 RNP loaded with either a non-targeting (NTC), a talin1-targeting (Tln1 gRNA), or a kindlin3-targeting (K3 gRNA) guide RNA. GAPDH served as loading control. ( C ) Densitometric quantification of talin1 and kindlin3 expression exemplarily shown in ( B ). N = 4 transfections. ( D , E ) Integrin β2 activation of the cells described in ( B ) assessed by FACS as relative mAb24 ( D ) and KIM127 ( E ) binding. Cells were either left untreated or treated with EDTA, TNFα, or PMA. N = 4 transfections. ( F ) Western blot analysis of Rap1, RIAM, Sharpin, RapL, Filamin A, and ILK expression in Hoxb8-derived neutrophil-like cells transfected with Cas9 RNP loaded with either a non-targeting RNA (NTC) or a guide RNA targeting Rap1, RIAM, Sharpin, RapL, Filamin A, or ILK. GAPDH served as loading control. ( G ) Densitometric quantification of Rap1, RIAM, Sharpin, RapL, Filamin A, and ILK expression exemplarily shown in ( F ). N = 6 transfections. ( H ) Integrin β2 activation of the cells described in ( F ) assessed by FACS as relative mAb24 binding. Cells were either untreated or treated with EDTA, TNFα, CXCL1, fMLP, or PMA. N = 6 transfections. The data points represent the results of the individual experiments. Values are given as mean ± SD. *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: CRISPR, Expressing, Control, Activity Assay, Western Blot, Derivative Assay, Transfection, Activation Assay, Binding Assay

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet:

Article Snippet: Antibody , anti-nAChR β2 (rabbit polyclonal) , Proteintech , Cat#: 17844–1-AP , WB (1:1000).

Techniques: Transfection, Construct, Control, Recombinant, Plasmid Preparation, Cloning, Software