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R&D Systems mouse ectolrp4 his tag

Figure 2. ColQ binds directly to the extracellular domain of LRP4. A, pull-down assays. Magnetic beads were conjugated with ColQ-Flag (+) or not (−) and were subsequently incubated with conditioned media (CM) of HEK 293T cells containing equal amounts of Myc-tagged ectodomain of LRP4 <t>(ectoLRP4-</t> Myc) or LRP6 (ectoLRP6-Myc) or with control medium (HEK 293T cells transfected with an empty vector). Precipitated proteins were analyzed by Western immunoblotting with anti-Myc antibodies. ColQ-Flag interacted with ectoLRP4-Myc but not with ectoLRP6-Myc. Inputs show that comparable amounts of ectoLRP4-Myc and ectoLRP6-Myc were incubated with the ColQ-coated beads; n = 3. B, same experiment as in (A) except that higher concentrations of ectoLRP6-Myc than ectoLRP4-Myc were tested. Even in these conditions, ColQ bound only to ectoLRP4 and not to ectoLRP6. C, uncoated (−) or ColQ-coated (+) beads were incubated with CM expressing ectoLRP4-AP or with ectoLRP4-AP puriﬁed from the CM. Puriﬁed ectoLRP4-AP bound to ColQ-Flag, indicating a direct interaction between ectoLRP4 and ColQ. D, magnetic beads conjugated with equal amounts of ectoLRP4-Myc and ectoLRP6-Myc were incubated with the same amount of enzymatic activity of puriﬁed AChE–ColQ (A12 asymmetric forms) or AChE (monomeric globular G1 forms). Bound AChE–ColQ or AChE was quantiﬁed by measuring AChE activity. Results are expressed as the mean ± SEM percentage normalized to the control value set as 100% (AChE– ColQ bound to control [CT] uncoated beads). n = 4; ****p < 0.0001, using two-way ANOVA followed by Tukey’s multiple comparison post hoc test. For

Mouse Ectolrp4 His Tag, supplied by R&D Systems, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "The collagen ColQ binds to LRP4 and regulates the activation of the Muscle-Specific Kinase-LRP4 receptor complex by agrin at the neuromuscular junction."

Article Title: The collagen ColQ binds to LRP4 and regulates the activation of the Muscle-Specific Kinase-LRP4 receptor complex by agrin at the neuromuscular junction.

Journal: The Journal of biological chemistry

doi: 10.1016/j.jbc.2023.104962

Figure Legend Snippet: Figure 2. ColQ binds directly to the extracellular domain of LRP4. A, pull-down assays. Magnetic beads were conjugated with ColQ-Flag (+) or not (−) and were subsequently incubated with conditioned media (CM) of HEK 293T cells containing equal amounts of Myc-tagged ectodomain of LRP4 (ectoLRP4- Myc) or LRP6 (ectoLRP6-Myc) or with control medium (HEK 293T cells transfected with an empty vector). Precipitated proteins were analyzed by Western immunoblotting with anti-Myc antibodies. ColQ-Flag interacted with ectoLRP4-Myc but not with ectoLRP6-Myc. Inputs show that comparable amounts of ectoLRP4-Myc and ectoLRP6-Myc were incubated with the ColQ-coated beads; n = 3. B, same experiment as in (A) except that higher concentrations of ectoLRP6-Myc than ectoLRP4-Myc were tested. Even in these conditions, ColQ bound only to ectoLRP4 and not to ectoLRP6. C, uncoated (−) or ColQ-coated (+) beads were incubated with CM expressing ectoLRP4-AP or with ectoLRP4-AP puriﬁed from the CM. Puriﬁed ectoLRP4-AP bound to ColQ-Flag, indicating a direct interaction between ectoLRP4 and ColQ. D, magnetic beads conjugated with equal amounts of ectoLRP4-Myc and ectoLRP6-Myc were incubated with the same amount of enzymatic activity of puriﬁed AChE–ColQ (A12 asymmetric forms) or AChE (monomeric globular G1 forms). Bound AChE–ColQ or AChE was quantiﬁed by measuring AChE activity. Results are expressed as the mean ± SEM percentage normalized to the control value set as 100% (AChE– ColQ bound to control [CT] uncoated beads). n = 4; ****p < 0.0001, using two-way ANOVA followed by Tukey’s multiple comparison post hoc test. For

Techniques Used: Magnetic Beads, Incubation, Control, Transfection, Plasmid Preparation, Western Blot, Expressing, Activity Assay, Comparison

Figure 3. Surface plasmon resonance analysis of ColQ–LRP4 interaction. A, sensorgram of the binding of ColQ-Flag to a sensor chip covalently coated with anti-Flag antibodies to obtain a sensor surface covered with ColQ. Binding responses were measured using report points 30 s after the beginning of the dissociation phase and are indicated in RUs (resonance units). The level of immobilized ColQ reached about 3900 RU. Reference surface was obtained by injecting preparations from untransfected cells (CT) at the same protein concentration as ColQ-Flag preparations. B, 750 nM of puriﬁed ectoLRP4 (blue curve) or puriﬁed ectoMuSK (red curve) were passed over the ColQ-coated and reference surfaces. Sensorgrams represent the differential speciﬁc binding to ColQ after subtraction of the signals obtained on the reference surface. In contrast to ectoLRP4 (binding response of 23 RU), there was no binding of ectoMuSK to ColQ. C, sensorgram of the single-cycle kinetics. Five increasing (1.5-fold) concentrations (296, 444, 666, 1000, and 1500 nM) of puriﬁed ectoLRP4 were sequentially loaded on the ColQ-coated or reference surfaces without any regeneration step. Reference surface and drift signals were subtracted to obtain the accurate binding proﬁles. The black curve overlaid on the experimental data (blue curve) was obtained by ﬁtting the binding proﬁles to a 1:1 binding model with drifting baseline. The association (kon) and dissociation (koff) constants were 9.64 ± 2.86 × 104 M−1 s−1 and 9.1 ± 2.89 × 10−3 s−1, respectively, corresponding to a Kd of 9.4 ± 0.19 × 10−8 M (n = 2). D, conditioned medium containing ectoLRP4-AP at 60 nM (ectoLRP4 CM) or a same dilution of a control conditioned medium (control CM) lacking ectoLRP4-AP, but containing AP at the same concentration, was injected over ColQ-coated and reference sur- faces. Sensorgrams display the differential speciﬁc binding to ColQ after subtraction of the signals obtained with the reference surface. The binding response for ectoLRP4 CM was substantially higher than for control CM (34 RU versus eight RU), revealing a speciﬁc binding of ectoLRP4 to ColQ. AP, alkaline phosphatase; CM, conditioned medium; ColQ, collagen Q; LRP4, low-density lipoprotein receptor–related protein 4; MuSK, muscle-speciﬁc kinase; RU, resonance unit.

Figure Legend Snippet: Figure 3. Surface plasmon resonance analysis of ColQ–LRP4 interaction. A, sensorgram of the binding of ColQ-Flag to a sensor chip covalently coated with anti-Flag antibodies to obtain a sensor surface covered with ColQ. Binding responses were measured using report points 30 s after the beginning of the dissociation phase and are indicated in RUs (resonance units). The level of immobilized ColQ reached about 3900 RU. Reference surface was obtained by injecting preparations from untransfected cells (CT) at the same protein concentration as ColQ-Flag preparations. B, 750 nM of puriﬁed ectoLRP4 (blue curve) or puriﬁed ectoMuSK (red curve) were passed over the ColQ-coated and reference surfaces. Sensorgrams represent the differential speciﬁc binding to ColQ after subtraction of the signals obtained on the reference surface. In contrast to ectoLRP4 (binding response of 23 RU), there was no binding of ectoMuSK to ColQ. C, sensorgram of the single-cycle kinetics. Five increasing (1.5-fold) concentrations (296, 444, 666, 1000, and 1500 nM) of puriﬁed ectoLRP4 were sequentially loaded on the ColQ-coated or reference surfaces without any regeneration step. Reference surface and drift signals were subtracted to obtain the accurate binding proﬁles. The black curve overlaid on the experimental data (blue curve) was obtained by ﬁtting the binding proﬁles to a 1:1 binding model with drifting baseline. The association (kon) and dissociation (koff) constants were 9.64 ± 2.86 × 104 M−1 s−1 and 9.1 ± 2.89 × 10−3 s−1, respectively, corresponding to a Kd of 9.4 ± 0.19 × 10−8 M (n = 2). D, conditioned medium containing ectoLRP4-AP at 60 nM (ectoLRP4 CM) or a same dilution of a control conditioned medium (control CM) lacking ectoLRP4-AP, but containing AP at the same concentration, was injected over ColQ-coated and reference sur- faces. Sensorgrams display the differential speciﬁc binding to ColQ after subtraction of the signals obtained with the reference surface. The binding response for ectoLRP4 CM was substantially higher than for control CM (34 RU versus eight RU), revealing a speciﬁc binding of ectoLRP4 to ColQ. AP, alkaline phosphatase; CM, conditioned medium; ColQ, collagen Q; LRP4, low-density lipoprotein receptor–related protein 4; MuSK, muscle-speciﬁc kinase; RU, resonance unit.

Techniques Used: SPR Assay, Binding Assay, Protein Concentration, Control, Concentration Assay, Injection

Figure 4. Comparison of ColQ binding to LRP4 and to MuSK. A, pull-down assays. Magnetic beads conjugated with ColQ-Flag (+) or not (−) were incubated with the same amounts of ectoLRP4-Myc or ectoMuSK-Myc as shown in the input. EctoLRP4-Myc but not ectoMuSK-Myc precipitated with ColQ- Flag; n = 3. B, same experiment as in (A) except that ColQ-coated beads were incubated with higher ectoMuSK-Myc than ectoLRP4-Myc concentrations. C, magnetic beads conjugated with equal amounts of ectoLRP4-Myc and ectoMuSK-Myc were incubated with the same amount of enzymatic activity of puriﬁed AChE–ColQ or AChE. Bound AChE–ColQ or AChE was quantiﬁed by measuring AChE activity. Results are expressed as the mean ± SEM percentage of the control value set as 100% (AChE–ColQ bound to CT ColQ-free beads). n = 4; **p < 0.01; ***p < 0.001, using two-way ANOVA followed by Tukey’s multiple comparison post hoc test. For interaction factor: F = 7.41, p = 0.0045; for CT versus ectoLRP4 versus ectoMuSK: F = 8.63, p = 0.0024; for AChE–ColQ versus AChE: F = 91.22, p < 0.0001. No signiﬁcant binding of AChE–ColQ to ectoMuSK was detected, whereas AChE–ColQ bound to ectoLRP4. D, coim- munoprecipitation experiments. HEK 293T cells were cotransfected with either ColQ-Flag and ectoLRP4-Myc or ColQ-Flag and ectoMuSK-Myc.

Figure Legend Snippet: Figure 4. Comparison of ColQ binding to LRP4 and to MuSK. A, pull-down assays. Magnetic beads conjugated with ColQ-Flag (+) or not (−) were incubated with the same amounts of ectoLRP4-Myc or ectoMuSK-Myc as shown in the input. EctoLRP4-Myc but not ectoMuSK-Myc precipitated with ColQ- Flag; n = 3. B, same experiment as in (A) except that ColQ-coated beads were incubated with higher ectoMuSK-Myc than ectoLRP4-Myc concentrations. C, magnetic beads conjugated with equal amounts of ectoLRP4-Myc and ectoMuSK-Myc were incubated with the same amount of enzymatic activity of puriﬁed AChE–ColQ or AChE. Bound AChE–ColQ or AChE was quantiﬁed by measuring AChE activity. Results are expressed as the mean ± SEM percentage of the control value set as 100% (AChE–ColQ bound to CT ColQ-free beads). n = 4; **p < 0.01; ***p < 0.001, using two-way ANOVA followed by Tukey’s multiple comparison post hoc test. For interaction factor: F = 7.41, p = 0.0045; for CT versus ectoLRP4 versus ectoMuSK: F = 8.63, p = 0.0024; for AChE–ColQ versus AChE: F = 91.22, p < 0.0001. No signiﬁcant binding of AChE–ColQ to ectoMuSK was detected, whereas AChE–ColQ bound to ectoLRP4. D, coim- munoprecipitation experiments. HEK 293T cells were cotransfected with either ColQ-Flag and ectoLRP4-Myc or ColQ-Flag and ectoMuSK-Myc.

Techniques Used: Comparison, Binding Assay, Magnetic Beads, Incubation, Activity Assay, Control

Figure 6. Role of the ColQ C-terminal domain in the ColQ–LRP4 interaction. A, 25 or 50 μg of a Flag-tagged peptide corresponding to the last 27 amino acids of the ColQ C-terminal domain (Flag-ColQ Cter [425–451]) or of a Flag-control peptide corresponding to the scrambled 425 to 451 sequence were used to coat magnetic beads, which were subsequently incubated with puriﬁed ectoLRP4-Myc. Pulled down ectoLRP4-Myc was analyzed by Western immunoblot with anti-Myc antibodies. EctoLRP4-Myc interacts with the Flag-ColQ Cter peptide but not with the corresponding scrambled peptide. The image is representative of three independent experiments. B, magnetic beads coated with similar amounts of ColQ-Flag and ColQΔCt-Flag or ColQ-free beads as a control were incubated with conditioned media of HEK 293T cells containing equal amounts of ectoLRP4-Myc, as shown in inputs, or with control medium. C, quantiﬁcation of ectoLRP4 bound to ColQ or ColQΔCt from data in (B) reveals a lower binding of ectoLRP4 to ColQΔCt than to ColQ, albeit statistically not signiﬁcant. Results were normalized to precipitated ColQ or ColQΔCt and are expressed as the mean ± SEM of ColQ condition set as 100%; n = 5. ColQ, collagen Q; HEK, human embryonic kidney cell line; LRP4, low-density lipoprotein receptor–related protein 4.

Figure Legend Snippet: Figure 6. Role of the ColQ C-terminal domain in the ColQ–LRP4 interaction. A, 25 or 50 μg of a Flag-tagged peptide corresponding to the last 27 amino acids of the ColQ C-terminal domain (Flag-ColQ Cter [425–451]) or of a Flag-control peptide corresponding to the scrambled 425 to 451 sequence were used to coat magnetic beads, which were subsequently incubated with puriﬁed ectoLRP4-Myc. Pulled down ectoLRP4-Myc was analyzed by Western immunoblot with anti-Myc antibodies. EctoLRP4-Myc interacts with the Flag-ColQ Cter peptide but not with the corresponding scrambled peptide. The image is representative of three independent experiments. B, magnetic beads coated with similar amounts of ColQ-Flag and ColQΔCt-Flag or ColQ-free beads as a control were incubated with conditioned media of HEK 293T cells containing equal amounts of ectoLRP4-Myc, as shown in inputs, or with control medium. C, quantiﬁcation of ectoLRP4 bound to ColQ or ColQΔCt from data in (B) reveals a lower binding of ectoLRP4 to ColQΔCt than to ColQ, albeit statistically not signiﬁcant. Results were normalized to precipitated ColQ or ColQΔCt and are expressed as the mean ± SEM of ColQ condition set as 100%; n = 5. ColQ, collagen Q; HEK, human embryonic kidney cell line; LRP4, low-density lipoprotein receptor–related protein 4.

Techniques Used: Control, Sequencing, Magnetic Beads, Incubation, Western Blot, Binding Assay

Figure 7. Domains of LRP4 interacting with ColQ. A, schematic representation of ectoLRP4-AP and its deletion mutants. B, plate-binding assays. Same concentrations of ectoLRP4-AP and of the indicated deletion mutants were added to ColQ-coated or CT wells. Bound proteins were quantiﬁed by measuring AP activity. Results are expressed as the mean ± SEM percentage of the control value set as 100% (ectoLRP4-AP bound to CT wells). n ≥6; **p < 0.01; ****p < 0.0001, using two-way ANOVA followed by Tukey’s multiple comparison post hoc test. For interaction factor: F = 18.07, p < 0.0001; for CT versus ColQ: F = 162, p < 0.0001; for the comparison of the different ectoLRP4 mutants: F = 21.01, p < 0.0001. The N-terminal region of LRP4 plays a crucial role in ColQ–LRP4 interaction as its deletion (ectoLRP4Δ1-AP) compromised binding to ColQ. Conversely, the N-terminal region alone (ectoLRP4Δ234-AP) bound to ColQ at the same level as ectoLRP4-AP. C, ColQ-coated wells were incubated with 25 nM of ectoLRP4-AP in the presence or not of 500 nM puriﬁed re- combinant neural agrin. Results are the mean ± SEM percentage of ectoLRP4-AP bound to ColQ wells in the absence of agrin (set as 100%; n = 9, ***p < 0.001, using one-sample t test). The binding of ecto-LRP4 to ColQ was reduced by more than 50% in the presence of agrin. D, pull-down assay where ColQ- coated beads were incubated with 500 nM of His-tagged recombinant neural agrin. Agrin signals were analyzed by Western immunoblot using antibodies against His-tag. No agrin was coprecipitated with ColQ. AP, alkaline phosphatase; ColQ, collagen Q; LRP4, low-density lipoprotein receptor–related protein 2; ns, not signiﬁcant.

Figure Legend Snippet: Figure 7. Domains of LRP4 interacting with ColQ. A, schematic representation of ectoLRP4-AP and its deletion mutants. B, plate-binding assays. Same concentrations of ectoLRP4-AP and of the indicated deletion mutants were added to ColQ-coated or CT wells. Bound proteins were quantiﬁed by measuring AP activity. Results are expressed as the mean ± SEM percentage of the control value set as 100% (ectoLRP4-AP bound to CT wells). n ≥6; **p < 0.01; ****p < 0.0001, using two-way ANOVA followed by Tukey’s multiple comparison post hoc test. For interaction factor: F = 18.07, p < 0.0001; for CT versus ColQ: F = 162, p < 0.0001; for the comparison of the different ectoLRP4 mutants: F = 21.01, p < 0.0001. The N-terminal region of LRP4 plays a crucial role in ColQ–LRP4 interaction as its deletion (ectoLRP4Δ1-AP) compromised binding to ColQ. Conversely, the N-terminal region alone (ectoLRP4Δ234-AP) bound to ColQ at the same level as ectoLRP4-AP. C, ColQ-coated wells were incubated with 25 nM of ectoLRP4-AP in the presence or not of 500 nM puriﬁed re- combinant neural agrin. Results are the mean ± SEM percentage of ectoLRP4-AP bound to ColQ wells in the absence of agrin (set as 100%; n = 9, ***p < 0.001, using one-sample t test). The binding of ecto-LRP4 to ColQ was reduced by more than 50% in the presence of agrin. D, pull-down assay where ColQ- coated beads were incubated with 500 nM of His-tagged recombinant neural agrin. Agrin signals were analyzed by Western immunoblot using antibodies against His-tag. No agrin was coprecipitated with ColQ. AP, alkaline phosphatase; ColQ, collagen Q; LRP4, low-density lipoprotein receptor–related protein 2; ns, not signiﬁcant.

Techniques Used: Binding Assay, Activity Assay, Control, Comparison, Incubation, Pull Down Assay, Recombinant, Western Blot

Recombinant:

Article Title: EWS-FLI-1 creates a cell surface microenvironment conducive to IGF signaling by inducing pappalysin-1.
Article Snippet: The following siRNAs were used: human pappalysin-1 siRNA SMARTpool (M-005130-02-0005, Dharmacon) and Non-Targeting siRNA Pool #2 (D-001206-14-05, Dharmacon). siRNA transfection was performed using LipofectamineTM RNAiMAX Transfection Reagent (Thermo Fisher). .. Recombinant pappalysin-1 protein (2487-ZNF-020) was purchased from R&D Systems. .. The stanniocalcin 1 and 2 cDNAs were cloned by RTPCR with a C-terminal FLAG tag, sequence-verified, and subcloned into pCDH1-puro lentiviral vector (System Biosciences).

Article Title: EWS-FLI-1 creates a cell surface microenvironment conducive to IGF signaling by inducing pappalysin-1
Article Snippet: The following siRNAs were used: human pappalysin-1 siRNA SMARTpool (M-005130-02-0005, Dharmacon) and Non-Targeting siRNA Pool #2 (D-001206-14-05, Dharmacon). siRNA transfection was performed using LipofectamineTM RNAiMAX Transfection Reagent (Thermo Fisher). .. Recombinant pappalysin-1 protein (2487-ZNF-020) was purchased from R&D Systems. .. The stanniocalcin 1 and 2 cDNAs were cloned by RT- PCR with a C-terminal FLAG tag, sequence-verified, and subcloned into pCDH1-puro lentiviral vector (System Biosciences).

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Journal: The Journal of biological chemistry

Article Title: The collagen ColQ binds to LRP4 and regulates the activation of the Muscle-Specific Kinase-LRP4 receptor complex by agrin at the neuromuscular junction.

doi: 10.1016/j.jbc.2023.104962

Figure Lengend Snippet: Figure 2. ColQ binds directly to the extracellular domain of LRP4. A, pull-down assays. Magnetic beads were conjugated with ColQ-Flag (+) or not (−) and were subsequently incubated with conditioned media (CM) of HEK 293T cells containing equal amounts of Myc-tagged ectodomain of LRP4 (ectoLRP4- Myc) or LRP6 (ectoLRP6-Myc) or with control medium (HEK 293T cells transfected with an empty vector). Precipitated proteins were analyzed by Western immunoblotting with anti-Myc antibodies. ColQ-Flag interacted with ectoLRP4-Myc but not with ectoLRP6-Myc. Inputs show that comparable amounts of ectoLRP4-Myc and ectoLRP6-Myc were incubated with the ColQ-coated beads; n = 3. B, same experiment as in (A) except that higher concentrations of ectoLRP6-Myc than ectoLRP4-Myc were tested. Even in these conditions, ColQ bound only to ectoLRP4 and not to ectoLRP6. C, uncoated (−) or ColQ-coated (+) beads were incubated with CM expressing ectoLRP4-AP or with ectoLRP4-AP puriﬁed from the CM. Puriﬁed ectoLRP4-AP bound to ColQ-Flag, indicating a direct interaction between ectoLRP4 and ColQ. D, magnetic beads conjugated with equal amounts of ectoLRP4-Myc and ectoLRP6-Myc were incubated with the same amount of enzymatic activity of puriﬁed AChE–ColQ (A12 asymmetric forms) or AChE (monomeric globular G1 forms). Bound AChE–ColQ or AChE was quantiﬁed by measuring AChE activity. Results are expressed as the mean ± SEM percentage normalized to the control value set as 100% (AChE– ColQ bound to control [CT] uncoated beads). n = 4; ****p < 0.0001, using two-way ANOVA followed by Tukey’s multiple comparison post hoc test. For

Article Snippet: Recombinant rat neural agrin, mouse ectoLRP4 His-tag, and mouse ectoMuSK Fc Chimera were from R&D Systems.

Techniques: Magnetic Beads, Incubation, Control, Transfection, Plasmid Preparation, Western Blot, Expressing, Activity Assay, Comparison

Journal: The Journal of biological chemistry

Article Title: The collagen ColQ binds to LRP4 and regulates the activation of the Muscle-Specific Kinase-LRP4 receptor complex by agrin at the neuromuscular junction.

doi: 10.1016/j.jbc.2023.104962

Figure Lengend Snippet: Figure 3. Surface plasmon resonance analysis of ColQ–LRP4 interaction. A, sensorgram of the binding of ColQ-Flag to a sensor chip covalently coated with anti-Flag antibodies to obtain a sensor surface covered with ColQ. Binding responses were measured using report points 30 s after the beginning of the dissociation phase and are indicated in RUs (resonance units). The level of immobilized ColQ reached about 3900 RU. Reference surface was obtained by injecting preparations from untransfected cells (CT) at the same protein concentration as ColQ-Flag preparations. B, 750 nM of puriﬁed ectoLRP4 (blue curve) or puriﬁed ectoMuSK (red curve) were passed over the ColQ-coated and reference surfaces. Sensorgrams represent the differential speciﬁc binding to ColQ after subtraction of the signals obtained on the reference surface. In contrast to ectoLRP4 (binding response of 23 RU), there was no binding of ectoMuSK to ColQ. C, sensorgram of the single-cycle kinetics. Five increasing (1.5-fold) concentrations (296, 444, 666, 1000, and 1500 nM) of puriﬁed ectoLRP4 were sequentially loaded on the ColQ-coated or reference surfaces without any regeneration step. Reference surface and drift signals were subtracted to obtain the accurate binding proﬁles. The black curve overlaid on the experimental data (blue curve) was obtained by ﬁtting the binding proﬁles to a 1:1 binding model with drifting baseline. The association (kon) and dissociation (koff) constants were 9.64 ± 2.86 × 104 M−1 s−1 and 9.1 ± 2.89 × 10−3 s−1, respectively, corresponding to a Kd of 9.4 ± 0.19 × 10−8 M (n = 2). D, conditioned medium containing ectoLRP4-AP at 60 nM (ectoLRP4 CM) or a same dilution of a control conditioned medium (control CM) lacking ectoLRP4-AP, but containing AP at the same concentration, was injected over ColQ-coated and reference sur- faces. Sensorgrams display the differential speciﬁc binding to ColQ after subtraction of the signals obtained with the reference surface. The binding response for ectoLRP4 CM was substantially higher than for control CM (34 RU versus eight RU), revealing a speciﬁc binding of ectoLRP4 to ColQ. AP, alkaline phosphatase; CM, conditioned medium; ColQ, collagen Q; LRP4, low-density lipoprotein receptor–related protein 4; MuSK, muscle-speciﬁc kinase; RU, resonance unit.

Article Snippet: Recombinant rat neural agrin, mouse ectoLRP4 His-tag, and mouse ectoMuSK Fc Chimera were from R&D Systems.

Techniques: SPR Assay, Binding Assay, Protein Concentration, Control, Concentration Assay, Injection

Journal: The Journal of biological chemistry

Article Title: The collagen ColQ binds to LRP4 and regulates the activation of the Muscle-Specific Kinase-LRP4 receptor complex by agrin at the neuromuscular junction.

doi: 10.1016/j.jbc.2023.104962

Figure Lengend Snippet: Figure 4. Comparison of ColQ binding to LRP4 and to MuSK. A, pull-down assays. Magnetic beads conjugated with ColQ-Flag (+) or not (−) were incubated with the same amounts of ectoLRP4-Myc or ectoMuSK-Myc as shown in the input. EctoLRP4-Myc but not ectoMuSK-Myc precipitated with ColQ- Flag; n = 3. B, same experiment as in (A) except that ColQ-coated beads were incubated with higher ectoMuSK-Myc than ectoLRP4-Myc concentrations. C, magnetic beads conjugated with equal amounts of ectoLRP4-Myc and ectoMuSK-Myc were incubated with the same amount of enzymatic activity of puriﬁed AChE–ColQ or AChE. Bound AChE–ColQ or AChE was quantiﬁed by measuring AChE activity. Results are expressed as the mean ± SEM percentage of the control value set as 100% (AChE–ColQ bound to CT ColQ-free beads). n = 4; **p < 0.01; ***p < 0.001, using two-way ANOVA followed by Tukey’s multiple comparison post hoc test. For interaction factor: F = 7.41, p = 0.0045; for CT versus ectoLRP4 versus ectoMuSK: F = 8.63, p = 0.0024; for AChE–ColQ versus AChE: F = 91.22, p < 0.0001. No signiﬁcant binding of AChE–ColQ to ectoMuSK was detected, whereas AChE–ColQ bound to ectoLRP4. D, coim- munoprecipitation experiments. HEK 293T cells were cotransfected with either ColQ-Flag and ectoLRP4-Myc or ColQ-Flag and ectoMuSK-Myc.

Article Snippet: Recombinant rat neural agrin, mouse ectoLRP4 His-tag, and mouse ectoMuSK Fc Chimera were from R&D Systems.

Techniques: Comparison, Binding Assay, Magnetic Beads, Incubation, Activity Assay, Control

Journal: The Journal of biological chemistry

Article Title: The collagen ColQ binds to LRP4 and regulates the activation of the Muscle-Specific Kinase-LRP4 receptor complex by agrin at the neuromuscular junction.

doi: 10.1016/j.jbc.2023.104962

Figure Lengend Snippet: Figure 6. Role of the ColQ C-terminal domain in the ColQ–LRP4 interaction. A, 25 or 50 μg of a Flag-tagged peptide corresponding to the last 27 amino acids of the ColQ C-terminal domain (Flag-ColQ Cter [425–451]) or of a Flag-control peptide corresponding to the scrambled 425 to 451 sequence were used to coat magnetic beads, which were subsequently incubated with puriﬁed ectoLRP4-Myc. Pulled down ectoLRP4-Myc was analyzed by Western immunoblot with anti-Myc antibodies. EctoLRP4-Myc interacts with the Flag-ColQ Cter peptide but not with the corresponding scrambled peptide. The image is representative of three independent experiments. B, magnetic beads coated with similar amounts of ColQ-Flag and ColQΔCt-Flag or ColQ-free beads as a control were incubated with conditioned media of HEK 293T cells containing equal amounts of ectoLRP4-Myc, as shown in inputs, or with control medium. C, quantiﬁcation of ectoLRP4 bound to ColQ or ColQΔCt from data in (B) reveals a lower binding of ectoLRP4 to ColQΔCt than to ColQ, albeit statistically not signiﬁcant. Results were normalized to precipitated ColQ or ColQΔCt and are expressed as the mean ± SEM of ColQ condition set as 100%; n = 5. ColQ, collagen Q; HEK, human embryonic kidney cell line; LRP4, low-density lipoprotein receptor–related protein 4.

Article Snippet: Recombinant rat neural agrin, mouse ectoLRP4 His-tag, and mouse ectoMuSK Fc Chimera were from R&D Systems.

Techniques: Control, Sequencing, Magnetic Beads, Incubation, Western Blot, Binding Assay

Journal: The Journal of biological chemistry

Article Title: The collagen ColQ binds to LRP4 and regulates the activation of the Muscle-Specific Kinase-LRP4 receptor complex by agrin at the neuromuscular junction.

doi: 10.1016/j.jbc.2023.104962

Figure Lengend Snippet: Figure 7. Domains of LRP4 interacting with ColQ. A, schematic representation of ectoLRP4-AP and its deletion mutants. B, plate-binding assays. Same concentrations of ectoLRP4-AP and of the indicated deletion mutants were added to ColQ-coated or CT wells. Bound proteins were quantiﬁed by measuring AP activity. Results are expressed as the mean ± SEM percentage of the control value set as 100% (ectoLRP4-AP bound to CT wells). n ≥6; **p < 0.01; ****p < 0.0001, using two-way ANOVA followed by Tukey’s multiple comparison post hoc test. For interaction factor: F = 18.07, p < 0.0001; for CT versus ColQ: F = 162, p < 0.0001; for the comparison of the different ectoLRP4 mutants: F = 21.01, p < 0.0001. The N-terminal region of LRP4 plays a crucial role in ColQ–LRP4 interaction as its deletion (ectoLRP4Δ1-AP) compromised binding to ColQ. Conversely, the N-terminal region alone (ectoLRP4Δ234-AP) bound to ColQ at the same level as ectoLRP4-AP. C, ColQ-coated wells were incubated with 25 nM of ectoLRP4-AP in the presence or not of 500 nM puriﬁed re- combinant neural agrin. Results are the mean ± SEM percentage of ectoLRP4-AP bound to ColQ wells in the absence of agrin (set as 100%; n = 9, ***p < 0.001, using one-sample t test). The binding of ecto-LRP4 to ColQ was reduced by more than 50% in the presence of agrin. D, pull-down assay where ColQ- coated beads were incubated with 500 nM of His-tagged recombinant neural agrin. Agrin signals were analyzed by Western immunoblot using antibodies against His-tag. No agrin was coprecipitated with ColQ. AP, alkaline phosphatase; ColQ, collagen Q; LRP4, low-density lipoprotein receptor–related protein 2; ns, not signiﬁcant.

Article Snippet: Recombinant rat neural agrin, mouse ectoLRP4 His-tag, and mouse ectoMuSK Fc Chimera were from R&D Systems.

Techniques: Binding Assay, Activity Assay, Control, Comparison, Incubation, Pull Down Assay, Recombinant, Western Blot

Figure 1. PAPPA was highly expressed in pregnancy-associated breast cancer (PABC) tissues and cells. (a) mRNA levels of PAPPA were significantly higher in pregnancy-associated breast cancer (PABC) tissues than in normal breast tissues (n = 30). (b) The expression of PAPPA in PABC tissues and normal breast tissues were measured by IHC staining (n = 3 patients, scale bar = 50 μm). (c) Western-blot was used to detect the protein level of PAPPA in PABC tissues and the adjacent normal tissues (n = 3 patients). (d) mRNA levels of PAPPA were examined by qRT-PCR in breast cancer cell lines (T47D, MCF-7, BT549, MDA- MB-231, and MDA-MB-468) and normal breast epithelial cell MCF-10A. (E) The protein level of PAPPA were detected by western blot in breast cancer cell lines (T47D, MCF-7, BT549, MDA-MB-231, and MDA-MB-468) and in normal breast epithelial cell MCF-10A. The data were analyzed using SPSS19.0 and Prism9 software. The data were presented at mean ± standard deviation. **P < 0.01, ***P < 0.001.

Journal: Bioengineered

Article Title: Pregnancy-associated plasma protein-A (PAPPA) promotes breast cancer progression.

doi: 10.1080/21655979.2021.2000724

Figure Lengend Snippet: Figure 1. PAPPA was highly expressed in pregnancy-associated breast cancer (PABC) tissues and cells. (a) mRNA levels of PAPPA were significantly higher in pregnancy-associated breast cancer (PABC) tissues than in normal breast tissues (n = 30). (b) The expression of PAPPA in PABC tissues and normal breast tissues were measured by IHC staining (n = 3 patients, scale bar = 50 μm). (c) Western-blot was used to detect the protein level of PAPPA in PABC tissues and the adjacent normal tissues (n = 3 patients). (d) mRNA levels of PAPPA were examined by qRT-PCR in breast cancer cell lines (T47D, MCF-7, BT549, MDA- MB-231, and MDA-MB-468) and normal breast epithelial cell MCF-10A. (E) The protein level of PAPPA were detected by western blot in breast cancer cell lines (T47D, MCF-7, BT549, MDA-MB-231, and MDA-MB-468) and in normal breast epithelial cell MCF-10A. The data were analyzed using SPSS19.0 and Prism9 software. The data were presented at mean ± standard deviation. **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant PAPPA protein (2487-ZNF-020) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Immunohistochemistry, Western Blot, Quantitative RT-PCR, Software, Standard Deviation

Figure 2. Overexpression of PAPPA promoted proliferation, migration and invasion of breast cancer cells. MDA-MB-231 and MCF7 cell lines with relatively low expression of PAPPA were selected for for overexpression study. Cells were transfected with empty vector and pcDNA-PAPPA overexpression plasmid. (a) Western-blot was used to examine the overexpression of PAPPA after pcDNA-PAPPA transfection. (b) CCK-8 proliferation assay showed that overexpressing PAPPA significantly promoted cell proliferation. (c) EdU incorporation assay showed that overexpression of PAPPA increased the percentage of EdU positive cells. (d, e) Overexpression of PAPPA increased the migration and invasion ability in MDA-MB-231 and MCF7 cells. (f) Overexpression of PAPPA increased the levels of N-cadherin and vimentin (EMT markers) and decreased the protein levels of E-cadherin (epithelial marker). The data were analyzed using SPSS19.0 and Prism9 software. The data were presented at mean ± standard deviation. **P < 0.01, ***P < 0.001.

Journal: Bioengineered

Article Title: Pregnancy-associated plasma protein-A (PAPPA) promotes breast cancer progression.

doi: 10.1080/21655979.2021.2000724

Figure Lengend Snippet: Figure 2. Overexpression of PAPPA promoted proliferation, migration and invasion of breast cancer cells. MDA-MB-231 and MCF7 cell lines with relatively low expression of PAPPA were selected for for overexpression study. Cells were transfected with empty vector and pcDNA-PAPPA overexpression plasmid. (a) Western-blot was used to examine the overexpression of PAPPA after pcDNA-PAPPA transfection. (b) CCK-8 proliferation assay showed that overexpressing PAPPA significantly promoted cell proliferation. (c) EdU incorporation assay showed that overexpression of PAPPA increased the percentage of EdU positive cells. (d, e) Overexpression of PAPPA increased the migration and invasion ability in MDA-MB-231 and MCF7 cells. (f) Overexpression of PAPPA increased the levels of N-cadherin and vimentin (EMT markers) and decreased the protein levels of E-cadherin (epithelial marker). The data were analyzed using SPSS19.0 and Prism9 software. The data were presented at mean ± standard deviation. **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant PAPPA protein (2487-ZNF-020) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Over Expression, Migration, Expressing, Transfection, Plasmid Preparation, Western Blot, CCK-8 Assay, Proliferation Assay, Marker, Software, Standard Deviation

Figure 3. Knocking down PAPPA inhibited the proliferation, migration and invasion of breast cancer cells. BT549 and MDA-MB-468 with high PAPPA expressions were selected for knockdown experiments. Cells were transfected with si-NC (control siRNA) and si-PAPPA (siRNA targeting PAPPA). (a) Western-blot was used to examine PAPPA level after si-PAPPA silencing. (b) CCK-8 proliferation assay showed that silencing PAPPA significantly suppressed cell proliferation. (c) EdU incorporation assay showed that silencing of PAPPA decreased the percentage of EdU positive cells. (d, e) Silencing PAPPA impaired the migration and invasion ability in MDA-MB-231 and MCF7 cells. (f) PAPPA knockdown decreased the levels of N-cadherin and vimentin (EMT markers) and increased the protein level of E-cadherin (epithelial marker). The data were analyzed using SPSS19.0 and Prism9 software. The data were presented at mean ± standard deviation. **P < 0.01, ***P < 0.001.

Journal: Bioengineered

Article Title: Pregnancy-associated plasma protein-A (PAPPA) promotes breast cancer progression.

doi: 10.1080/21655979.2021.2000724

Figure Lengend Snippet: Figure 3. Knocking down PAPPA inhibited the proliferation, migration and invasion of breast cancer cells. BT549 and MDA-MB-468 with high PAPPA expressions were selected for knockdown experiments. Cells were transfected with si-NC (control siRNA) and si-PAPPA (siRNA targeting PAPPA). (a) Western-blot was used to examine PAPPA level after si-PAPPA silencing. (b) CCK-8 proliferation assay showed that silencing PAPPA significantly suppressed cell proliferation. (c) EdU incorporation assay showed that silencing of PAPPA decreased the percentage of EdU positive cells. (d, e) Silencing PAPPA impaired the migration and invasion ability in MDA-MB-231 and MCF7 cells. (f) PAPPA knockdown decreased the levels of N-cadherin and vimentin (EMT markers) and increased the protein level of E-cadherin (epithelial marker). The data were analyzed using SPSS19.0 and Prism9 software. The data were presented at mean ± standard deviation. **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant PAPPA protein (2487-ZNF-020) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Migration, Knockdown, Transfection, Control, Western Blot, CCK-8 Assay, Proliferation Assay, Marker, Software, Standard Deviation

Figure 4. Migratory capability of breast cancer cells is enhanced by co-culture with PAPPA-rich serum. (a) The level of PAPPA in serum of PABC patients and healthy subjects were detected by ELISA. (b-e). MDA-MB-231 and MCF7 were cultured with healthy control serum (CS), PABC patient serum (PS) and PS in the presence of PAPPA-antagonizing antibody (PS+anti- PAPPA). (b) CCK-8 proliferation assay revealed that PABC serum treatment enhanced cell proliferation, which was partially inhibited by PAPPA antibody. (c) Wound-healing migration assay and (d) transwell invasion assay showed that PABC serum treatment promoted cell migration and invasion ability, which partially inhibited by PAPPA antibody. (e) PABC serum treatment upregulated EMT markers (N-cadherin and vimentin) and PAPPA antibody antagonized this effect. The data were analyzed using SPSS19.0 and Prism9 software. The data were presented at mean ± standard deviation. **P < 0.01, ***P < 0.001. (* indicates comparison between CS and PS; # indicates comparison between PS and PS+anti-PAPPA).

Journal: Bioengineered

Article Title: Pregnancy-associated plasma protein-A (PAPPA) promotes breast cancer progression.

doi: 10.1080/21655979.2021.2000724

Figure Lengend Snippet: Figure 4. Migratory capability of breast cancer cells is enhanced by co-culture with PAPPA-rich serum. (a) The level of PAPPA in serum of PABC patients and healthy subjects were detected by ELISA. (b-e). MDA-MB-231 and MCF7 were cultured with healthy control serum (CS), PABC patient serum (PS) and PS in the presence of PAPPA-antagonizing antibody (PS+anti- PAPPA). (b) CCK-8 proliferation assay revealed that PABC serum treatment enhanced cell proliferation, which was partially inhibited by PAPPA antibody. (c) Wound-healing migration assay and (d) transwell invasion assay showed that PABC serum treatment promoted cell migration and invasion ability, which partially inhibited by PAPPA antibody. (e) PABC serum treatment upregulated EMT markers (N-cadherin and vimentin) and PAPPA antibody antagonized this effect. The data were analyzed using SPSS19.0 and Prism9 software. The data were presented at mean ± standard deviation. **P < 0.01, ***P < 0.001. (* indicates comparison between CS and PS; # indicates comparison between PS and PS+anti-PAPPA).

Article Snippet: Recombinant PAPPA protein (2487-ZNF-020) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Control, CCK-8 Assay, Proliferation Assay, Migration, Transwell Invasion Assay, Software, Standard Deviation, Comparison

Figure 5. MiR-497-5p negatively targeted PAPPA. (a) Venn Diagram showed that 14 miRNAs targeting PAPPA were predicted by Starbase, TargetScan, miRD and Tarbase database. (b) Cells were transfected with miRNA mimics, only miR-497-5p mimic reduced PAPPA mRNA levels. (c) Protein level of PAPPA was decreased by transfection with miR-497-5p mimic. (d) Expression of miR-497-5p was measured in PABC tissue and normal breast tissues by qRT-PCR. (e) Pearson correlation analysis between level of miR-497-5p and PAPPA in PABC cancer tissue. (f) Expression of miR-497-5p was measured in the serum of PABC patients and healthy subjects by qRT-PCR. (g) Pearson correlation analysis between level of miR-497-5p and PAPPA in PABC serum. (h). Expression of miR-497-5p was measured in breast cancer cell lines (T47D, MCF-7, BT549, MDA-MB-231 and MDA-MB-468) and in normal breast epithelial cell MCF-10A by qRT-PCR. The data were analyzed using SPSS19.0 and Prism9 software. The data were presented at mean ± standard deviation. **P < 0.01, ***P < 0.001.

Journal: Bioengineered

Article Title: Pregnancy-associated plasma protein-A (PAPPA) promotes breast cancer progression.

doi: 10.1080/21655979.2021.2000724

Figure Lengend Snippet: Figure 5. MiR-497-5p negatively targeted PAPPA. (a) Venn Diagram showed that 14 miRNAs targeting PAPPA were predicted by Starbase, TargetScan, miRD and Tarbase database. (b) Cells were transfected with miRNA mimics, only miR-497-5p mimic reduced PAPPA mRNA levels. (c) Protein level of PAPPA was decreased by transfection with miR-497-5p mimic. (d) Expression of miR-497-5p was measured in PABC tissue and normal breast tissues by qRT-PCR. (e) Pearson correlation analysis between level of miR-497-5p and PAPPA in PABC cancer tissue. (f) Expression of miR-497-5p was measured in the serum of PABC patients and healthy subjects by qRT-PCR. (g) Pearson correlation analysis between level of miR-497-5p and PAPPA in PABC serum. (h). Expression of miR-497-5p was measured in breast cancer cell lines (T47D, MCF-7, BT549, MDA-MB-231 and MDA-MB-468) and in normal breast epithelial cell MCF-10A by qRT-PCR. The data were analyzed using SPSS19.0 and Prism9 software. The data were presented at mean ± standard deviation. **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant PAPPA protein (2487-ZNF-020) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Software, Standard Deviation

Figure 6. Overexpression of PAPPA rescued the inhibitory effect of miR-497-5p on the proliferation, migration and invasion of breast cancer cells. (a) CCK-8 proliferation assay revealed that transfection of MDA-MB-231 and MCF7 with miR-497-5p mimic inhibited cell proliferation, which was rescued by the co-transfection of pcDNA-PAPPA overexpression plasmid. (b) Wound-healing migration assay and (c) transwell invasion assay further showed that miR-497-5p mimic also inhibited cell migration and invasion ability, which was rescued by PAPPA overexpression. (d) miR-497-5p mimic downregulated EMT markers (N-cadherin and vimentin) and upregulated E-Cadherin, and PAPPA overexpression antagonized the effect. The data were analyzed using SPSS19.0 and Prism9 software. The data were presented at mean ± standard deviation. **P < 0.01, ***P < 0.001. (* indicates comparison between miR-NC and miR-497- 5p; # indicates comparison between miR-497-5p and miR-497-5p+pcDNA-PAPPA).

Journal: Bioengineered

Article Title: Pregnancy-associated plasma protein-A (PAPPA) promotes breast cancer progression.

doi: 10.1080/21655979.2021.2000724

Figure Lengend Snippet: Figure 6. Overexpression of PAPPA rescued the inhibitory effect of miR-497-5p on the proliferation, migration and invasion of breast cancer cells. (a) CCK-8 proliferation assay revealed that transfection of MDA-MB-231 and MCF7 with miR-497-5p mimic inhibited cell proliferation, which was rescued by the co-transfection of pcDNA-PAPPA overexpression plasmid. (b) Wound-healing migration assay and (c) transwell invasion assay further showed that miR-497-5p mimic also inhibited cell migration and invasion ability, which was rescued by PAPPA overexpression. (d) miR-497-5p mimic downregulated EMT markers (N-cadherin and vimentin) and upregulated E-Cadherin, and PAPPA overexpression antagonized the effect. The data were analyzed using SPSS19.0 and Prism9 software. The data were presented at mean ± standard deviation. **P < 0.01, ***P < 0.001. (* indicates comparison between miR-NC and miR-497- 5p; # indicates comparison between miR-497-5p and miR-497-5p+pcDNA-PAPPA).

Article Snippet: Recombinant PAPPA protein (2487-ZNF-020) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Over Expression, Migration, CCK-8 Assay, Proliferation Assay, Transfection, Cotransfection, Plasmid Preparation, Transwell Invasion Assay, Software, Standard Deviation, Comparison

Figure 7. PAPPA promoted tumorigenesis and metastasis of breast cancer cells in vivo. A total number of 10 female immunodeficient nude mice were subcutaneously injected with MDA-MB-231 cells. The mice were randomly divided into two groups (5 mice in each group): vector group (injected with PBS every three days) and p-PAPPA group (injected with recombinant p-PAPPA protein every three days). The injection of PAPPA significantly promoted the tumor growth (a) and tumor weight (b). (c) IHC staining of the cell proliferation marker Ki-67 and PAPPA in the tumor sections of the vector and p-PAPPA group (scale bar = 100 μm). (d) Hematoxylin and Eosin (HE) staining of lung tissue revealed increased number of metastatic nodules in the lung tissue in the group with PAPPA treatment (scale bar = 400 μm). The data were analyzed using SPSS19.0 and Prism9 software. The data were presented at mean ± standard deviation. **P < 0.01, ***P < 0.001.

Journal: Bioengineered

Article Title: Pregnancy-associated plasma protein-A (PAPPA) promotes breast cancer progression.

doi: 10.1080/21655979.2021.2000724

Figure Lengend Snippet: Figure 7. PAPPA promoted tumorigenesis and metastasis of breast cancer cells in vivo. A total number of 10 female immunodeficient nude mice were subcutaneously injected with MDA-MB-231 cells. The mice were randomly divided into two groups (5 mice in each group): vector group (injected with PBS every three days) and p-PAPPA group (injected with recombinant p-PAPPA protein every three days). The injection of PAPPA significantly promoted the tumor growth (a) and tumor weight (b). (c) IHC staining of the cell proliferation marker Ki-67 and PAPPA in the tumor sections of the vector and p-PAPPA group (scale bar = 100 μm). (d) Hematoxylin and Eosin (HE) staining of lung tissue revealed increased number of metastatic nodules in the lung tissue in the group with PAPPA treatment (scale bar = 400 μm). The data were analyzed using SPSS19.0 and Prism9 software. The data were presented at mean ± standard deviation. **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant PAPPA protein (2487-ZNF-020) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: In Vivo, Injection, Plasmid Preparation, Recombinant, Immunohistochemistry, Marker, Staining, Software, Standard Deviation

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1) Product Images from "The collagen ColQ binds to LRP4 and regulates the activation of the Muscle-Specific Kinase-LRP4 receptor complex by agrin at the neuromuscular junction."

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