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R&D Systems polyclonal sheep anti mouse pear1
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Polyclonal Sheep Anti Mouse Pear1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+pear1/pmc10842823-282-14-18?v=R%26D+Systems
Average 91 stars, based on 3 article reviews

polyclonal sheep anti mouse pear1 - by Bioz Stars, 2026-08

91/100 stars

Images

1) Product Images from "Jedi-1/MEGF12-mediated phagocytosis controls the pro-neurogenic properties of microglia in the ventricular-subventricular zone"

Article Title: Jedi-1/MEGF12-mediated phagocytosis controls the pro-neurogenic properties of microglia in the ventricular-subventricular zone

Journal: Cell reports

doi: 10.1016/j.celrep.2023.113423

Figure Legend Snippet: KEY RESOURCES TABLE

Techniques Used: Recombinant, Marker, In Situ, RNA Sequencing, Knock-Out, Mutagenesis, Real-time Polymerase Chain Reaction, Software

Image Search Results

Figure 2. PEAR1 exacerbates TNBC cell metastasis. (A) MDA-MB-231 cells with stable PEAR1 knockdown (shPEAR1) or PEAR1 overexpression (oe-PEAR1) were generated and verified as shown in Supplemental Figure 2A. Nontargeting shRNA was used as a negative control for shPEAR1 (shnc). The corresponding empty vector (Vector) was used as a negative control for oe-PEAR1. Quantification of invasive shPEAR1- and oe-PEAR1–treated MDA-MB-231 cells in a Transwell assay; quantification of the relative migration area of shPEAR1- and oe-PEAR1–treated MDA-MB-231 cells in the wound healing assay; and viability curves of shPEAR1- and oe-PEAR1–treated MDA-MB-231 cells generated with a CCK-8 assay kit (n = 3; mean ± SEM). (B) Quantification of invasive shPEAR1- and oe-PEAR1–treated SUM159 cells in a Transwell assay; quantification of the relative migration area of shPEAR1- and oe-PEAR1–treated SUM159 cells in the wound healing assay; and viability curves of shPEAR1- and oe-PEAR1–treated SUM159 cells generated with a CCK-8 assay kit (n = 3; mean ± SEM). (C) Quantification of invasive oe-PEAR1– treated MDA-MB-468 cells in a Transwell assay; relative migration area of oe-PEAR1–treated MDA-MB-468 cells quantified using the wound healing assay; and viability of oe-PEAR1–treated MDA-MB-468 cells assessed by a CCK-8 assay kit (n = 3; mean ± SEM). (D) Quantification of metastatic foci in the lungs and livers of nude mice i.v. injected with shPEAR1 and shnc MDA-MB-231 cells by H&E staining (n = 5 mice per group; mean ± SEM). Unpaired 2-tailed t tests were used for A–D; 1-way ANOVA followed by Dunnett’s test was used for A and B. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Journal: Journal of Clinical Investigation

Article Title: LOXL2-induced PEAR1 Ser891 phosphorylation suppresses CD44 degradation and promotes triple-negative breast cancer metastasis

doi: 10.1172/jci177357

Figure Lengend Snippet: Figure 2. PEAR1 exacerbates TNBC cell metastasis. (A) MDA-MB-231 cells with stable PEAR1 knockdown (shPEAR1) or PEAR1 overexpression (oe-PEAR1) were generated and verified as shown in Supplemental Figure 2A. Nontargeting shRNA was used as a negative control for shPEAR1 (shnc). The corresponding empty vector (Vector) was used as a negative control for oe-PEAR1. Quantification of invasive shPEAR1- and oe-PEAR1–treated MDA-MB-231 cells in a Transwell assay; quantification of the relative migration area of shPEAR1- and oe-PEAR1–treated MDA-MB-231 cells in the wound healing assay; and viability curves of shPEAR1- and oe-PEAR1–treated MDA-MB-231 cells generated with a CCK-8 assay kit (n = 3; mean ± SEM). (B) Quantification of invasive shPEAR1- and oe-PEAR1–treated SUM159 cells in a Transwell assay; quantification of the relative migration area of shPEAR1- and oe-PEAR1–treated SUM159 cells in the wound healing assay; and viability curves of shPEAR1- and oe-PEAR1–treated SUM159 cells generated with a CCK-8 assay kit (n = 3; mean ± SEM). (C) Quantification of invasive oe-PEAR1– treated MDA-MB-468 cells in a Transwell assay; relative migration area of oe-PEAR1–treated MDA-MB-468 cells quantified using the wound healing assay; and viability of oe-PEAR1–treated MDA-MB-468 cells assessed by a CCK-8 assay kit (n = 3; mean ± SEM). (D) Quantification of metastatic foci in the lungs and livers of nude mice i.v. injected with shPEAR1 and shnc MDA-MB-231 cells by H&E staining (n = 5 mice per group; mean ± SEM). Unpaired 2-tailed t tests were used for A–D; 1-way ANOVA followed by Dunnett’s test was used for A and B. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: For endogenous co-IP, the lysates were incubated with the anti-PEAR1 antibody (developed as described above) or anti-CD44 antibody (15675-1-AP, Proteintech) under gentle rotation overnight at 4°C, while corresponding isotype IgG (5415&2729, Cell Signaling Technology) was used as a negative control.

Techniques: Knockdown, Over Expression, Generated, shRNA, Negative Control, Plasmid Preparation, Transwell Assay, Migration, Wound Healing Assay, CCK-8 Assay, Injection, Staining

Figure 7. The LOXL2/PEAR1/CD44 axis is upregulated in TNBC and is associated with poor overall survival. (A) Quantification of PEAR1, phos- pho-PEAR1 Ser891, LOXL2, and CD44 staining scores by IHC in TNBC and corresponding adjacent samples (n = 80 for TAT and tumor samples; mean ± SEM). (B) Overall survival of patients with TNBC based on PEAR1, phospho-PEAR1 (Ser891), LOXL2, and CD44 expression levels (n values as indicated; log-rank test). (C) Heatmap of the correlation between the expression levels of PEAR1, phospho-PEAR1 Ser891, LOXL2, and CD44 in TNBC samples (n = 80; Pearson’s correlation analysis). (D) The prognostic effect of the risk score of phospho-PEAR1 Ser891 and its combination with CD44 for patients with TNBC (time-dependent ROC curve analysis). (E) Schematic diagram of the mechanisms by which the LOXL2/PEAR1/CD44 pathway regulates TNBC metastasis. Unpaired 2-tailed t tests were used for A; log-rank test was used for B; Pearson’s correlation analysis was used for C; time-depen- dent ROC curve analysis was used for D. ****P < 0.0001.

Journal: Journal of Clinical Investigation

Article Title: LOXL2-induced PEAR1 Ser891 phosphorylation suppresses CD44 degradation and promotes triple-negative breast cancer metastasis

doi: 10.1172/jci177357

Figure Lengend Snippet: Figure 7. The LOXL2/PEAR1/CD44 axis is upregulated in TNBC and is associated with poor overall survival. (A) Quantification of PEAR1, phos- pho-PEAR1 Ser891, LOXL2, and CD44 staining scores by IHC in TNBC and corresponding adjacent samples (n = 80 for TAT and tumor samples; mean ± SEM). (B) Overall survival of patients with TNBC based on PEAR1, phospho-PEAR1 (Ser891), LOXL2, and CD44 expression levels (n values as indicated; log-rank test). (C) Heatmap of the correlation between the expression levels of PEAR1, phospho-PEAR1 Ser891, LOXL2, and CD44 in TNBC samples (n = 80; Pearson’s correlation analysis). (D) The prognostic effect of the risk score of phospho-PEAR1 Ser891 and its combination with CD44 for patients with TNBC (time-dependent ROC curve analysis). (E) Schematic diagram of the mechanisms by which the LOXL2/PEAR1/CD44 pathway regulates TNBC metastasis. Unpaired 2-tailed t tests were used for A; log-rank test was used for B; Pearson’s correlation analysis was used for C; time-depen- dent ROC curve analysis was used for D. ****P < 0.0001.

Techniques: Staining, Expressing

( A ) Schematic of CD44-interacting proteins identified using IP-MS. CD44 and its interacting proteins were immunoprecipitated from whole-cell lysates of MDA-MB-231 cells using an anti-CD44 antibody and identified using MS. The IgG isotype served as a negative control. Specific proteins that interact with CD44 were screened. These proteins were detected in anti-CD44 IP samples but not in IgG IP samples; or the relative abundance ratio was greater than 200 in anti-CD44 IP samples compared to that in IgG-IP samples (area CD44/area IgG). ( B ) Bubble plots of the top 10 pathways obtained from KEGG enrichment analyses. ( C ) The top 20 effective proteins obtained from MS sorting according to the confidence score. ( D ) Co-IP of whole-cell lysates of MDA-MB-231 cells with anti-PEAR1 and anti-CD44 antibodies. The IgG isotype was used as a negative control. Results are representative of 3 independent experiments. ( E ) Representative IF staining of MDA-MB-231 cells with anti-PEAR1 (green) and anti-CD44 (red) antibodies and nuclei (blue). Scale bars: 20 μm (original image) and 10 μm (enlarged image). Quantitative analysis of the rate of PEAR1 and CD44 colocalization. ( F ) Representative IHC staining of the tissue microarray containing breast cancer and adjacent tissue samples with an anti-PEAR1 antibody. Scale bars: 50 μm. The PEAR1 staining scores were quantified as indicated ( n = 86 for TATs, n = 126 for tumors; mean ± SEM). ( G ) Total overall survival of patients with breast cancer and with TNBC based on PEAR1 expression level ( n values as indicated; log-rank test). KEGG pathway enrichment analyses were performed using the online tools DAVID and KOBAS ( B ); unpaired 2-tailed t tests were used for F ; log-rank test was used for G . **** P < 0.0001.

Journal: The Journal of Clinical Investigation

Article Title: LOXL2-induced PEAR1 Ser891 phosphorylation suppresses CD44 degradation and promotes triple-negative breast cancer metastasis

doi: 10.1172/JCI177357

Figure Lengend Snippet: ( A ) Schematic of CD44-interacting proteins identified using IP-MS. CD44 and its interacting proteins were immunoprecipitated from whole-cell lysates of MDA-MB-231 cells using an anti-CD44 antibody and identified using MS. The IgG isotype served as a negative control. Specific proteins that interact with CD44 were screened. These proteins were detected in anti-CD44 IP samples but not in IgG IP samples; or the relative abundance ratio was greater than 200 in anti-CD44 IP samples compared to that in IgG-IP samples (area CD44/area IgG). ( B ) Bubble plots of the top 10 pathways obtained from KEGG enrichment analyses. ( C ) The top 20 effective proteins obtained from MS sorting according to the confidence score. ( D ) Co-IP of whole-cell lysates of MDA-MB-231 cells with anti-PEAR1 and anti-CD44 antibodies. The IgG isotype was used as a negative control. Results are representative of 3 independent experiments. ( E ) Representative IF staining of MDA-MB-231 cells with anti-PEAR1 (green) and anti-CD44 (red) antibodies and nuclei (blue). Scale bars: 20 μm (original image) and 10 μm (enlarged image). Quantitative analysis of the rate of PEAR1 and CD44 colocalization. ( F ) Representative IHC staining of the tissue microarray containing breast cancer and adjacent tissue samples with an anti-PEAR1 antibody. Scale bars: 50 μm. The PEAR1 staining scores were quantified as indicated ( n = 86 for TATs, n = 126 for tumors; mean ± SEM). ( G ) Total overall survival of patients with breast cancer and with TNBC based on PEAR1 expression level ( n values as indicated; log-rank test). KEGG pathway enrichment analyses were performed using the online tools DAVID and KOBAS ( B ); unpaired 2-tailed t tests were used for F ; log-rank test was used for G . **** P < 0.0001.

Article Snippet: An anti–phospho-PEAR1 Ser891 antibody (p-PEAR1 Ser891 Ab) was generated by Shanghai GL Biochem Co. Rabbit antiserum against PEAR1 Ser891 phosphorylation was generated using the peptide Cys-RGSSRLDRSY(pS)YSYSNGP coupled to the carrier protein keyhole limpet hemocyanin (KLH), the Ser891 of which is phosphorylated and indicated as pS.

Techniques: Protein-Protein interactions, Immunoprecipitation, Negative Control, Co-Immunoprecipitation Assay, Staining, Immunohistochemistry, Microarray, Expressing

( A ) MDA-MB-231 cells with stable PEAR1 knockdown (shPEAR1) or PEAR1 overexpression (oe-PEAR1) were generated and verified as shown in . Nontargeting shRNA was used as a negative control for shPEAR1 (shnc). The corresponding empty vector (Vector) was used as a negative control for oe-PEAR1. Quantification of invasive shPEAR1- and oe-PEAR1–treated MDA-MB-231 cells in a Transwell assay; quantification of the relative migration area of shPEAR1- and oe-PEAR1–treated MDA-MB-231 cells in the wound healing assay; and viability curves of shPEAR1- and oe-PEAR1–treated MDA-MB-231 cells generated with a CCK-8 assay kit ( n = 3; mean ± SEM). ( B ) Quantification of invasive shPEAR1- and oe-PEAR1–treated SUM159 cells in a Transwell assay; quantification of the relative migration area of shPEAR1- and oe-PEAR1–treated SUM159 cells in the wound healing assay; and viability curves of shPEAR1- and oe-PEAR1–treated SUM159 cells generated with a CCK-8 assay kit ( n = 3; mean ± SEM). ( C ) Quantification of invasive oe-PEAR1–treated MDA-MB-468 cells in a Transwell assay; relative migration area of oe-PEAR1–treated MDA-MB-468 cells quantified using the wound healing assay; and viability of oe-PEAR1–treated MDA-MB-468 cells assessed by a CCK-8 assay kit ( n = 3; mean ± SEM). ( D ) Quantification of metastatic foci in the lungs and livers of nude mice i.v. injected with shPEAR1 and shnc MDA-MB-231 cells by H&E staining ( n = 5 mice per group; mean ± SEM). Unpaired 2-tailed t tests were used for A – D ; 1-way ANOVA followed by Dunnett’s test was used for A and B . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Journal: The Journal of Clinical Investigation

Article Title: LOXL2-induced PEAR1 Ser891 phosphorylation suppresses CD44 degradation and promotes triple-negative breast cancer metastasis

doi: 10.1172/JCI177357

Figure Lengend Snippet: ( A ) MDA-MB-231 cells with stable PEAR1 knockdown (shPEAR1) or PEAR1 overexpression (oe-PEAR1) were generated and verified as shown in . Nontargeting shRNA was used as a negative control for shPEAR1 (shnc). The corresponding empty vector (Vector) was used as a negative control for oe-PEAR1. Quantification of invasive shPEAR1- and oe-PEAR1–treated MDA-MB-231 cells in a Transwell assay; quantification of the relative migration area of shPEAR1- and oe-PEAR1–treated MDA-MB-231 cells in the wound healing assay; and viability curves of shPEAR1- and oe-PEAR1–treated MDA-MB-231 cells generated with a CCK-8 assay kit ( n = 3; mean ± SEM). ( B ) Quantification of invasive shPEAR1- and oe-PEAR1–treated SUM159 cells in a Transwell assay; quantification of the relative migration area of shPEAR1- and oe-PEAR1–treated SUM159 cells in the wound healing assay; and viability curves of shPEAR1- and oe-PEAR1–treated SUM159 cells generated with a CCK-8 assay kit ( n = 3; mean ± SEM). ( C ) Quantification of invasive oe-PEAR1–treated MDA-MB-468 cells in a Transwell assay; relative migration area of oe-PEAR1–treated MDA-MB-468 cells quantified using the wound healing assay; and viability of oe-PEAR1–treated MDA-MB-468 cells assessed by a CCK-8 assay kit ( n = 3; mean ± SEM). ( D ) Quantification of metastatic foci in the lungs and livers of nude mice i.v. injected with shPEAR1 and shnc MDA-MB-231 cells by H&E staining ( n = 5 mice per group; mean ± SEM). Unpaired 2-tailed t tests were used for A – D ; 1-way ANOVA followed by Dunnett’s test was used for A and B . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Techniques: Knockdown, Over Expression, Generated, shRNA, Negative Control, Plasmid Preparation, Transwell Assay, Migration, Wound Healing Assay, CCK-8 Assay, Injection, Staining

( A ) Co-IP of whole-cell lysates of HEK293T cells transiently transfected with PEAR1-ICD–Flag and CD44-ICD–HA plasmids with anti-Flag and anti-HA agarose beads. Cells transfected with the corresponding empty vector plasmids were used as negative controls. ( B ) The interaction between PEAR1-ECD and CD44-ECD was detected using pulldown with anti-His Dynabeads in a solution containing purified PEAR1-ECD–His and CD44-ECD–Fc protein. Fc was used as a negative control. ( C ) Quantification of the mammosphere diameters and quantities of shPEAR1 and oe-PEAR1 MDA-MB-231 cells ( n = 3; mean ± SEM). ( D ) Expression levels of CD44, OCT4, SOX2, NANOG, E-cadherin, and vimentin in total lysates and of CD44-ICD in nuclear proteins of shPEAR1 and oe-PEAR1 MDA-MB-231 cells as determined by Western blotting analysis. Quantitative analysis of the relative gray values of full-length CD44 with GAPDH and nuclear CD44-ICD with histone H3 ( n = 3; mean ± SD). ( E ) CD44 mRNA expression levels in shPEAR1 and oe-PEAR1 MDA-MB-231 cells were determined by RT-qPCR. The diagram shows the relative expression of mRNAs normalized to that of 18S rRNA ( n = 3; mean ± SD). ( F and G ) Western blotting of CD44 ( F ) and IF staining of CD44 (red), LAMP1 (green), and nuclei (blue) ( G ) in shPEAR1 MDA-MB-231 cells treated with or without dynasore, SGC-AAK-1, pitstop 2, or MG132 for 1 hour. β-Actin served as an internal control ( F ). Number of endocytotic CD44 foci per cell and mean intensity of CD44 ( G ). Scale bars: 20 μm. One-way ANOVA followed by Dunnett’s test was used for C – E and G ; unpaired 2-tailed t tests were used for C – E . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The Western blotting results are representative of 3 independent experiments.

Journal: The Journal of Clinical Investigation

Article Title: LOXL2-induced PEAR1 Ser891 phosphorylation suppresses CD44 degradation and promotes triple-negative breast cancer metastasis

doi: 10.1172/JCI177357

Figure Lengend Snippet: ( A ) Co-IP of whole-cell lysates of HEK293T cells transiently transfected with PEAR1-ICD–Flag and CD44-ICD–HA plasmids with anti-Flag and anti-HA agarose beads. Cells transfected with the corresponding empty vector plasmids were used as negative controls. ( B ) The interaction between PEAR1-ECD and CD44-ECD was detected using pulldown with anti-His Dynabeads in a solution containing purified PEAR1-ECD–His and CD44-ECD–Fc protein. Fc was used as a negative control. ( C ) Quantification of the mammosphere diameters and quantities of shPEAR1 and oe-PEAR1 MDA-MB-231 cells ( n = 3; mean ± SEM). ( D ) Expression levels of CD44, OCT4, SOX2, NANOG, E-cadherin, and vimentin in total lysates and of CD44-ICD in nuclear proteins of shPEAR1 and oe-PEAR1 MDA-MB-231 cells as determined by Western blotting analysis. Quantitative analysis of the relative gray values of full-length CD44 with GAPDH and nuclear CD44-ICD with histone H3 ( n = 3; mean ± SD). ( E ) CD44 mRNA expression levels in shPEAR1 and oe-PEAR1 MDA-MB-231 cells were determined by RT-qPCR. The diagram shows the relative expression of mRNAs normalized to that of 18S rRNA ( n = 3; mean ± SD). ( F and G ) Western blotting of CD44 ( F ) and IF staining of CD44 (red), LAMP1 (green), and nuclei (blue) ( G ) in shPEAR1 MDA-MB-231 cells treated with or without dynasore, SGC-AAK-1, pitstop 2, or MG132 for 1 hour. β-Actin served as an internal control ( F ). Number of endocytotic CD44 foci per cell and mean intensity of CD44 ( G ). Scale bars: 20 μm. One-way ANOVA followed by Dunnett’s test was used for C – E and G ; unpaired 2-tailed t tests were used for C – E . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The Western blotting results are representative of 3 independent experiments.

Techniques: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Purification, Negative Control, Expressing, Western Blot, Quantitative RT-PCR, Staining, Control

$( A ) PEAR1 was immunoprecipitated from MDA-MB-231 cell lysates with anti-PEAR1 antibody, after which the levels of serine/threonine phosphorylation or tyrosine phosphorylation were measured with Western blotting. IgG served as an isotype control. ( B ) Serine/threonine phosphorylation levels of PEAR1 in HEK293T cells transiently transfected with oe-PEAR1–WT-Flag, oe-PEAR1–All SA-Flag, oe-PEAR1–S795A-Flag, oe-PEAR1–S891A-Flag, oe-PEAR1–S953A-Flag, oe-PEAR1–S976A-Flag, or oe-PEAR1–S1029A-Flag plasmids. “All SA” indicates that all serine was mutated to alanine at all 5 residues. ( C ) PEAR1 serine/threonine phosphorylation levels in MDA-MB-231 cells stably expressing PEAR1-WT-Flag, SA-Flag, or S891A-Flag. ( D ) The interaction between PEAR1 and CD44 in MDA-MB-231 cells was detected by co-IP with anti-PEAR1 or anti-CD44 antibodies. ( E ) CD44, OCT4, SOX2, NANOG, E-cadherin, vimentin, and CD44-ICD protein expression in total lysates and nuclear fractions of the indicated MDA-MB-231 cells. ( F and G ) Western blotting of CD44 ( F ) and IF staining of CD44 (red), LAMP1 (green), and nuclei (blue) ( G ) in oe-PEAR1–WT-Flag and oe-PEAR1–S891A-Flag MDA-MB-231 cells treated with or without dynasore, SGC-AAK-1, pitstop 2, or MG132 for 1 hour. Number of endocytotic CD44 foci per cell and mean intensity of CD44. Scale bars: 20 μm. ( H and I ) Quantification of mammosphere diameters and quantities ( H ); and quantification of invasive cells in Transwell assay and relative migration area in wound healing assay ( I ) of the indicated MDA-MB-231 cells ( n = 3; mean ± SEM). ( J ) Quantification of metastatic foci by H&E staining in the lungs and livers of nude mice i.v. injected with the indicated MDA-MB-231 cells ( n = 5 mice per group; mean ± SEM). One-way ANOVA followed by Dunnett’s test was used for G – J . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The Western blotting results are representative of 3 independent experiments.$

Journal: The Journal of Clinical Investigation

Article Title: LOXL2-induced PEAR1 Ser891 phosphorylation suppresses CD44 degradation and promotes triple-negative breast cancer metastasis

doi: 10.1172/JCI177357

Figure Lengend Snippet: ( A ) PEAR1 was immunoprecipitated from MDA-MB-231 cell lysates with anti-PEAR1 antibody, after which the levels of serine/threonine phosphorylation or tyrosine phosphorylation were measured with Western blotting. IgG served as an isotype control. ( B ) Serine/threonine phosphorylation levels of PEAR1 in HEK293T cells transiently transfected with oe-PEAR1–WT-Flag, oe-PEAR1–All SA-Flag, oe-PEAR1–S795A-Flag, oe-PEAR1–S891A-Flag, oe-PEAR1–S953A-Flag, oe-PEAR1–S976A-Flag, or oe-PEAR1–S1029A-Flag plasmids. “All SA” indicates that all serine was mutated to alanine at all 5 residues. ( C ) PEAR1 serine/threonine phosphorylation levels in MDA-MB-231 cells stably expressing PEAR1-WT-Flag, SA-Flag, or S891A-Flag. ( D ) The interaction between PEAR1 and CD44 in MDA-MB-231 cells was detected by co-IP with anti-PEAR1 or anti-CD44 antibodies. ( E ) CD44, OCT4, SOX2, NANOG, E-cadherin, vimentin, and CD44-ICD protein expression in total lysates and nuclear fractions of the indicated MDA-MB-231 cells. ( F and G ) Western blotting of CD44 ( F ) and IF staining of CD44 (red), LAMP1 (green), and nuclei (blue) ( G ) in oe-PEAR1–WT-Flag and oe-PEAR1–S891A-Flag MDA-MB-231 cells treated with or without dynasore, SGC-AAK-1, pitstop 2, or MG132 for 1 hour. Number of endocytotic CD44 foci per cell and mean intensity of CD44. Scale bars: 20 μm. ( H and I ) Quantification of mammosphere diameters and quantities ( H ); and quantification of invasive cells in Transwell assay and relative migration area in wound healing assay ( I ) of the indicated MDA-MB-231 cells ( n = 3; mean ± SEM). ( J ) Quantification of metastatic foci by H&E staining in the lungs and livers of nude mice i.v. injected with the indicated MDA-MB-231 cells ( n = 5 mice per group; mean ± SEM). One-way ANOVA followed by Dunnett’s test was used for G – J . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The Western blotting results are representative of 3 independent experiments.

Techniques: Immunoprecipitation, Phospho-proteomics, Western Blot, Control, Transfection, Stable Transfection, Expressing, Co-Immunoprecipitation Assay, Staining, Transwell Assay, Migration, Wound Healing Assay, Injection

( A ) The interaction between LOXL2 and PEAR1 was detected by pulldown with Dynabeads in the supernatant of MDA-MB-231 cells, to which 15 μg exogenous PEAR1-ECD-his was added. ( B ) LOXL2 levels in the supernatants of various TNBC cell lines were quantified by ELISA (MDA-MB-231, red; MDA-MB-468, orange; SUM159, yellow). The complete culture medium served as a negative control (blue) ( n = 2; mean ± SD). ( C and D ) Quantification of invasive cells in Transwell assay; and relative migration area in wound healing assay of MDA-MB-231 cells treated with 0, 5, or 10 ng/mL LOXL2 ( n = 3; mean ± SEM). ( E ) Phosphorylated PEAR1, full-length CD44, and CD44-ICD levels were detected in MDA-MB-231 cells and SUM159 cells treated with 0, 5, 10, 20, or 50 ng/mL LOXL2. ( F ) Schematic showing the structures of full-length LOXL2 protein, the LOXL2-SRCR1-3 truncation, and the LOXL2-SRCR1-2 truncation. ( G – I ) Quantification of invasive cells in Transwell assay and relative migration area in wound healing assay ( n = 3; mean ± SEM) and detection of PEAR1 phosphorylation levels with Western blotting in MDA-MB-231 cells treated with 0.2 nM full-length LOXL2 or its truncation construct. ( J ) Interaction of PEAR1-ECD–His with LOXL2 in the supernatant of MDA-MB-231 cells was inhibited by 20 μg/mL simtuzumab. ( K and L ) Quantification of invasive cells in Transwell assay and relative migration area in wound healing assay in MDA-MB-231 cells treated with 0, 5, 10, or 20 μg/mL simtuzumab ( n = 3; mean ± SEM). ( M ) Levels of phosphorylated PEAR1, CD44, and CD44-ICD were determined in MDA-MB-231 cells treated with 0, 5, 10, or 20 μg/mL simtuzumab. One-way ANOVA followed by Dunnett’s test was used for C , D , G , H , K , and L . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The Western blotting results are representative of 3 independent experiments.

Journal: The Journal of Clinical Investigation

Article Title: LOXL2-induced PEAR1 Ser891 phosphorylation suppresses CD44 degradation and promotes triple-negative breast cancer metastasis

doi: 10.1172/JCI177357

Figure Lengend Snippet: ( A ) The interaction between LOXL2 and PEAR1 was detected by pulldown with Dynabeads in the supernatant of MDA-MB-231 cells, to which 15 μg exogenous PEAR1-ECD-his was added. ( B ) LOXL2 levels in the supernatants of various TNBC cell lines were quantified by ELISA (MDA-MB-231, red; MDA-MB-468, orange; SUM159, yellow). The complete culture medium served as a negative control (blue) ( n = 2; mean ± SD). ( C and D ) Quantification of invasive cells in Transwell assay; and relative migration area in wound healing assay of MDA-MB-231 cells treated with 0, 5, or 10 ng/mL LOXL2 ( n = 3; mean ± SEM). ( E ) Phosphorylated PEAR1, full-length CD44, and CD44-ICD levels were detected in MDA-MB-231 cells and SUM159 cells treated with 0, 5, 10, 20, or 50 ng/mL LOXL2. ( F ) Schematic showing the structures of full-length LOXL2 protein, the LOXL2-SRCR1-3 truncation, and the LOXL2-SRCR1-2 truncation. ( G – I ) Quantification of invasive cells in Transwell assay and relative migration area in wound healing assay ( n = 3; mean ± SEM) and detection of PEAR1 phosphorylation levels with Western blotting in MDA-MB-231 cells treated with 0.2 nM full-length LOXL2 or its truncation construct. ( J ) Interaction of PEAR1-ECD–His with LOXL2 in the supernatant of MDA-MB-231 cells was inhibited by 20 μg/mL simtuzumab. ( K and L ) Quantification of invasive cells in Transwell assay and relative migration area in wound healing assay in MDA-MB-231 cells treated with 0, 5, 10, or 20 μg/mL simtuzumab ( n = 3; mean ± SEM). ( M ) Levels of phosphorylated PEAR1, CD44, and CD44-ICD were determined in MDA-MB-231 cells treated with 0, 5, 10, or 20 μg/mL simtuzumab. One-way ANOVA followed by Dunnett’s test was used for C , D , G , H , K , and L . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The Western blotting results are representative of 3 independent experiments.

Techniques: Enzyme-linked Immunosorbent Assay, Negative Control, Transwell Assay, Migration, Wound Healing Assay, Phospho-proteomics, Western Blot, Construct

( A ) Schematic showing the structural features of PEAR1. ( B ) The interaction between LOXL2 and various PEAR1-ECD domain peptides was detected using ELISA ( n = 2; mean ± SD). ( C – E ) Quantification of invasive cells in Transwell assay; quantification of relative migration area in wound healing assay ( n = 3; mean ± SEM); detection of phosphorylated PEAR1, CD44, and CD44-ICD using Western blotting of MDA-MB-231 cells treated with or without 10 ng/mL LOXL2 and 1 or 5 μg/mL PEAR1-EMI domain protein. EMI del, EMI deletion. ( F – H ) Quantification of invasive cells in Transwell assay; quantification of relative migration area in wound healing assay ( n = 3; mean ± SEM); and detection of CD44 and CD44-ICD by Western blotting of MDA-MB-231 cells with oe-PEAR1-EMI domain deficiency. EMI del, EMI deletion. ( I – K ) Quantification of invasive cells in Transwell assay; quantification of relative migration area in wound healing assay ( n = 3; mean ± SEM); and detection of phosphorylated PEAR1, CD44, and CD44-ICD using Western blotting of MDA-MB-231 cells treated with 10 ng/mL LOXL2 and 5 or 10 μg/mL PEAR1 Fab-HSA. Quantitative analysis of the gray values of phospho–PEAR1 Ser/Thr to PEAR1, CD44 to GAPDH, and CD44-ICD to histone H3 ( n = 3; mean ± SD). ( L ) The number of metastatic foci in the lung and liver was significantly reduced by i.v. injection of PEAR1 Fab-HSA at a dose of 3.35 mg/kg for 40 days in a mouse model of metastasis generated by i.v. injection of MDA-MB-231 cells ( n = 5 mice per group; mean ± SEM). One-way ANOVA followed by Dunnett’s test was used for C , D , F , G , and I – L . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The Western blotting results are representative of 3 independent experiments.

Journal: The Journal of Clinical Investigation

Article Title: LOXL2-induced PEAR1 Ser891 phosphorylation suppresses CD44 degradation and promotes triple-negative breast cancer metastasis

doi: 10.1172/JCI177357

Figure Lengend Snippet: ( A ) Schematic showing the structural features of PEAR1. ( B ) The interaction between LOXL2 and various PEAR1-ECD domain peptides was detected using ELISA ( n = 2; mean ± SD). ( C – E ) Quantification of invasive cells in Transwell assay; quantification of relative migration area in wound healing assay ( n = 3; mean ± SEM); detection of phosphorylated PEAR1, CD44, and CD44-ICD using Western blotting of MDA-MB-231 cells treated with or without 10 ng/mL LOXL2 and 1 or 5 μg/mL PEAR1-EMI domain protein. EMI del, EMI deletion. ( F – H ) Quantification of invasive cells in Transwell assay; quantification of relative migration area in wound healing assay ( n = 3; mean ± SEM); and detection of CD44 and CD44-ICD by Western blotting of MDA-MB-231 cells with oe-PEAR1-EMI domain deficiency. EMI del, EMI deletion. ( I – K ) Quantification of invasive cells in Transwell assay; quantification of relative migration area in wound healing assay ( n = 3; mean ± SEM); and detection of phosphorylated PEAR1, CD44, and CD44-ICD using Western blotting of MDA-MB-231 cells treated with 10 ng/mL LOXL2 and 5 or 10 μg/mL PEAR1 Fab-HSA. Quantitative analysis of the gray values of phospho–PEAR1 Ser/Thr to PEAR1, CD44 to GAPDH, and CD44-ICD to histone H3 ( n = 3; mean ± SD). ( L ) The number of metastatic foci in the lung and liver was significantly reduced by i.v. injection of PEAR1 Fab-HSA at a dose of 3.35 mg/kg for 40 days in a mouse model of metastasis generated by i.v. injection of MDA-MB-231 cells ( n = 5 mice per group; mean ± SEM). One-way ANOVA followed by Dunnett’s test was used for C , D , F , G , and I – L . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The Western blotting results are representative of 3 independent experiments.

Techniques: Enzyme-linked Immunosorbent Assay, Transwell Assay, Migration, Wound Healing Assay, Western Blot, Injection, Generated

( A ) Quantification of PEAR1, phospho-PEAR1 Ser891, LOXL2, and CD44 staining scores by IHC in TNBC and corresponding adjacent samples ( n = 80 for TAT and tumor samples; mean ± SEM). ( B ) Overall survival of patients with TNBC based on PEAR1, phospho-PEAR1 (Ser891), LOXL2, and CD44 expression levels ( n values as indicated; log-rank test). ( C ) Heatmap of the correlation between the expression levels of PEAR1, phospho-PEAR1 Ser891, LOXL2, and CD44 in TNBC samples ( n = 80; Pearson’s correlation analysis). ( D ) The prognostic effect of the risk score of phospho-PEAR1 Ser891 and its combination with CD44 for patients with TNBC (time-dependent ROC curve analysis). ( E ) Schematic diagram of the mechanisms by which the LOXL2/PEAR1/CD44 pathway regulates TNBC metastasis. Unpaired 2-tailed t tests were used for A ; log-rank test was used for B ; Pearson’s correlation analysis was used for C ; time-dependent ROC curve analysis was used for D . **** P < 0.0001.

Journal: The Journal of Clinical Investigation

Article Title: LOXL2-induced PEAR1 Ser891 phosphorylation suppresses CD44 degradation and promotes triple-negative breast cancer metastasis

doi: 10.1172/JCI177357

Figure Lengend Snippet: ( A ) Quantification of PEAR1, phospho-PEAR1 Ser891, LOXL2, and CD44 staining scores by IHC in TNBC and corresponding adjacent samples ( n = 80 for TAT and tumor samples; mean ± SEM). ( B ) Overall survival of patients with TNBC based on PEAR1, phospho-PEAR1 (Ser891), LOXL2, and CD44 expression levels ( n values as indicated; log-rank test). ( C ) Heatmap of the correlation between the expression levels of PEAR1, phospho-PEAR1 Ser891, LOXL2, and CD44 in TNBC samples ( n = 80; Pearson’s correlation analysis). ( D ) The prognostic effect of the risk score of phospho-PEAR1 Ser891 and its combination with CD44 for patients with TNBC (time-dependent ROC curve analysis). ( E ) Schematic diagram of the mechanisms by which the LOXL2/PEAR1/CD44 pathway regulates TNBC metastasis. Unpaired 2-tailed t tests were used for A ; log-rank test was used for B ; Pearson’s correlation analysis was used for C ; time-dependent ROC curve analysis was used for D . **** P < 0.0001.

Techniques: Staining, Expressing

Journal: Cell reports

Article Title: Jedi-1/MEGF12-mediated phagocytosis controls the pro-neurogenic properties of microglia in the ventricular-subventricular zone

doi: 10.1016/j.celrep.2023.113423

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For frozen sections, the following primary antibodies were used (with specific diluents and dilutions): Polyclonal Sheep anti-Mouse PEAR1 (R&D Systems, Cat#AF7607; RRID: AB_2943660; used at 1:50 in blocking solution); Polyclonal Rabbit anti-Mouse IBA1 (same as for paraffin section but used at 1:500 in blocking solution); Monoclonal Rat anti-Mouse CD68 (Bio-Rad, Cat#MCA1957; RRID: AB_322219; used at 1:100 in blocking solution).

Techniques: Recombinant, Marker, In Situ, RNA Sequencing, Knock-Out, Mutagenesis, Real-time Polymerase Chain Reaction, Software

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