sortilin  (Alomone Labs)


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    Alomone Labs sortilin
    Loss of <t>sortilin</t> aggravates TrkC and TrkB phenotypes in p75 NTR knockout mice. a , Quantification of proprioceptive neurons in L4/L5 DRGs using markers for NF200 and TrkC (n=8). b , Reduction of muscle spindles in hind-limbs in P1 Sort1 −/−
    Sortilin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sortilin/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sortilin - by Bioz Stars, 2022-05
    93/100 stars

    Images

    1) Product Images from "Sortilin associates with Trk receptors to enhance anterograde transport and signaling by neurotrophins"

    Article Title: Sortilin associates with Trk receptors to enhance anterograde transport and signaling by neurotrophins

    Journal: Nature neuroscience

    doi: 10.1038/nn.2689

    Loss of sortilin aggravates TrkC and TrkB phenotypes in p75 NTR knockout mice. a , Quantification of proprioceptive neurons in L4/L5 DRGs using markers for NF200 and TrkC (n=8). b , Reduction of muscle spindles in hind-limbs in P1 Sort1 −/−
    Figure Legend Snippet: Loss of sortilin aggravates TrkC and TrkB phenotypes in p75 NTR knockout mice. a , Quantification of proprioceptive neurons in L4/L5 DRGs using markers for NF200 and TrkC (n=8). b , Reduction of muscle spindles in hind-limbs in P1 Sort1 −/−

    Techniques Used: Knock-Out, Mouse Assay

    Absent sortilin expression induces TrkA phenotypes in Ntrk1 +/− mice. a , Schematic representation of possible genotypes in offspring from Sort1 +/− / Ntrk1 +/− and Sort1 −/− / Ntrk1 +/+ breeding. b , Genotype frequency of
    Figure Legend Snippet: Absent sortilin expression induces TrkA phenotypes in Ntrk1 +/− mice. a , Schematic representation of possible genotypes in offspring from Sort1 +/− / Ntrk1 +/− and Sort1 −/− / Ntrk1 +/+ breeding. b , Genotype frequency of

    Techniques Used: Expressing, Mouse Assay

    Endogenous sortilin facilitates TrkA trafficking and signaling in neurons. a , Movement of EGFP-TrkA in neurites of cultured DRG neurons. Arrows indicate anterograde (red) and retrograde (yellow) movement of vesicles. b , Kymograph of a neurite as shown
    Figure Legend Snippet: Endogenous sortilin facilitates TrkA trafficking and signaling in neurons. a , Movement of EGFP-TrkA in neurites of cultured DRG neurons. Arrows indicate anterograde (red) and retrograde (yellow) movement of vesicles. b , Kymograph of a neurite as shown

    Techniques Used: Cell Culture

    Sortilin facilitates Trk signaling in neurons. a , WB of phospho-ERK1/2 (MAPK) in primary DRG neuron cultures after stimulation with NGF for 10 min. b , Quantification of ERK1/2 activation from 5 experiments as depicted in panel a. c , TUJ-1 staining of
    Figure Legend Snippet: Sortilin facilitates Trk signaling in neurons. a , WB of phospho-ERK1/2 (MAPK) in primary DRG neuron cultures after stimulation with NGF for 10 min. b , Quantification of ERK1/2 activation from 5 experiments as depicted in panel a. c , TUJ-1 staining of

    Techniques Used: Western Blot, Activation Assay, Staining

    Abnormal gait and peripheral neuropathy in sortilin and p75 NTR double knockout mice a , Abnormal hind-limb posture in Sort −/− / Ngfr −/− mice. b , Correlation between genotype and penetrance of the various phenotypes. Parenthesis
    Figure Legend Snippet: Abnormal gait and peripheral neuropathy in sortilin and p75 NTR double knockout mice a , Abnormal hind-limb posture in Sort −/− / Ngfr −/− mice. b , Correlation between genotype and penetrance of the various phenotypes. Parenthesis

    Techniques Used: Double Knockout, Mouse Assay

    Sortilin deficiency aggravates TrkA phenotypes in p75 NTR knockout mice. a , Thermal response as measured by Hargreaves test (n=10). b , Quantification of nociceptive neuron subtypes in L4-L5 DRG (n=8). c , Electron micrographs showing the morphology of Remak
    Figure Legend Snippet: Sortilin deficiency aggravates TrkA phenotypes in p75 NTR knockout mice. a , Thermal response as measured by Hargreaves test (n=10). b , Quantification of nociceptive neuron subtypes in L4-L5 DRG (n=8). c , Electron micrographs showing the morphology of Remak

    Techniques Used: Knock-Out, Mouse Assay

    Sortilin interacts with Trk receptors. a , Co-localization of sortilin with p75 NTR and Trk receptors in DRG neurons. b , Left: co-immunoprecipitation of sortilin and TrkA in HEK293 using pan-anti-Trk or anti-sortilin antibody, respectively (IP), Middle:
    Figure Legend Snippet: Sortilin interacts with Trk receptors. a , Co-localization of sortilin with p75 NTR and Trk receptors in DRG neurons. b , Left: co-immunoprecipitation of sortilin and TrkA in HEK293 using pan-anti-Trk or anti-sortilin antibody, respectively (IP), Middle:

    Techniques Used: Immunoprecipitation

    2) Product Images from "Nerve growth factor and its receptor tyrosine kinase TrkA are overexpressed in cervical squamous cell carcinoma, et al. Nerve growth factor and its receptor tyrosine kinase TrkA are overexpressed in cervical squamous cell carcinoma"

    Article Title: Nerve growth factor and its receptor tyrosine kinase TrkA are overexpressed in cervical squamous cell carcinoma, et al. Nerve growth factor and its receptor tyrosine kinase TrkA are overexpressed in cervical squamous cell carcinoma

    Journal: FASEB BioAdvances

    doi: 10.1096/fba.2020-00016

    Detection of nerves in the microenvironment of cervical cancer. Immunohistochemical detection of nerves was performed using the pan‐neuronal marker PGP9.5 in a cohort of 287 cervical cancers and 30 normal cervical tissue samples. (A‐D) Nerves stained for PGP9.5 adjacent to stroma (St) and cancer cells (CC). Immunohistochemical detection of proNGF (E), NGF (F), TrkA (G), p75 NTR (H) and sortilin (I) was performed in serial sections containing the same nerve shown in (D). Scale bars: 50 μm
    Figure Legend Snippet: Detection of nerves in the microenvironment of cervical cancer. Immunohistochemical detection of nerves was performed using the pan‐neuronal marker PGP9.5 in a cohort of 287 cervical cancers and 30 normal cervical tissue samples. (A‐D) Nerves stained for PGP9.5 adjacent to stroma (St) and cancer cells (CC). Immunohistochemical detection of proNGF (E), NGF (F), TrkA (G), p75 NTR (H) and sortilin (I) was performed in serial sections containing the same nerve shown in (D). Scale bars: 50 μm

    Techniques Used: Immunohistochemistry, Marker, Staining

    Sortilin expression in cervical cancers and normal cervical tissue. Immunohistochemical detection of sortilin in normal cervical tissues (A), adenocarcinomas (AC) (B) and squamous cell carcinomas (SCC) (C, D). Scale bars: 50 μm (A‐D). (E) Digital quantification of sortilin expression was performed and h‐scores were calculated. Data are expressed as medians (horizontal line in the center of the box) and box limits indicate the interquartile range (IQR) with the whiskers extending 1.5 times the IQR from the 25th and 75th percentiles; outliers are represented by dots. * P
    Figure Legend Snippet: Sortilin expression in cervical cancers and normal cervical tissue. Immunohistochemical detection of sortilin in normal cervical tissues (A), adenocarcinomas (AC) (B) and squamous cell carcinomas (SCC) (C, D). Scale bars: 50 μm (A‐D). (E) Digital quantification of sortilin expression was performed and h‐scores were calculated. Data are expressed as medians (horizontal line in the center of the box) and box limits indicate the interquartile range (IQR) with the whiskers extending 1.5 times the IQR from the 25th and 75th percentiles; outliers are represented by dots. * P

    Techniques Used: Expressing, Immunohistochemistry

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    Alomone Labs anti sortilin antibody
    Frequency distribution of <t>sortilin</t> levels Sortilin levels (0 = no staining, 1 = low intensity staining, 2 = intermediate intensity staining, 3 = high intensity staining) were measured in breast cancers and normal breast tissues. A. Distribution in normal tissues versus breast tumors. B. Distribution in invasive lobular carcinomas (ILC) versus invasive ductal carcinomas (IDC). C. Distribution in lymph node negative (LN-) versus lymph node positive (LN+) cancers. Number of cases (n) is indicated. Statistical significance of the difference between groups are reported in Table 1 .
    Anti Sortilin Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sortilin antibody/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti sortilin antibody - by Bioz Stars, 2022-05
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    Frequency distribution of sortilin levels Sortilin levels (0 = no staining, 1 = low intensity staining, 2 = intermediate intensity staining, 3 = high intensity staining) were measured in breast cancers and normal breast tissues. A. Distribution in normal tissues versus breast tumors. B. Distribution in invasive lobular carcinomas (ILC) versus invasive ductal carcinomas (IDC). C. Distribution in lymph node negative (LN-) versus lymph node positive (LN+) cancers. Number of cases (n) is indicated. Statistical significance of the difference between groups are reported in Table 1 .

    Journal: Oncotarget

    Article Title: Sortilin is associated with breast cancer aggressiveness and contributes to tumor cell adhesion and invasion

    doi:

    Figure Lengend Snippet: Frequency distribution of sortilin levels Sortilin levels (0 = no staining, 1 = low intensity staining, 2 = intermediate intensity staining, 3 = high intensity staining) were measured in breast cancers and normal breast tissues. A. Distribution in normal tissues versus breast tumors. B. Distribution in invasive lobular carcinomas (ILC) versus invasive ductal carcinomas (IDC). C. Distribution in lymph node negative (LN-) versus lymph node positive (LN+) cancers. Number of cases (n) is indicated. Statistical significance of the difference between groups are reported in Table 1 .

    Article Snippet: The efficiency of sortilin knockdown was assessed by Western-blotting using anti-sortilin antibody (ANT009, Alomone Labs, Israel).

    Techniques: Staining

    Impact of sortilin knockdown on migration and invasion of breast cancer cells A. Scratch assay. Breast cancer cells (MDA-MB-231, SKBR-3 and MCF-7) were transfected with siRNA against sortilin (siSORT) and universal negative control siRNA (siCONT). Scratching of the cell layer was performed 48 h after transfection and reduction in gap area was measured over 6 h. SiSORT inhibited migration only in MDA-MB-231 cells. B. Transwell invasion assay of MDA-MB-231 cells. Transwell assays were set up 48 h after siRNA transfection and cells were allowed to invade for 48 h. To take under account a potential impact of cell adhesion on the assay, cells were counted on both sides of the Transwell filter. Left panel, white columns represent the number of cells on the upper side of the filter, and the black columns the number of cells on the down side. Right panel, the percentage of invading cells in siSORT versus siCONT is represented. C. Western-blot detection of vimentin and activation of SRC, FAK, Akt and Erk1/2, 72 h after transfection with siSORT versus siCONT. Antibodies against vimentin, β-actin, SRC, phospho-SRC (Tyr416), FAK, phospho-FAK (Tyr576/577), Akt, phosphor-Akt (Ser473), Erk1/2, phosphor-Erk1/2 were used. For panel A and B, error bars represent SD. * p

    Journal: Oncotarget

    Article Title: Sortilin is associated with breast cancer aggressiveness and contributes to tumor cell adhesion and invasion

    doi:

    Figure Lengend Snippet: Impact of sortilin knockdown on migration and invasion of breast cancer cells A. Scratch assay. Breast cancer cells (MDA-MB-231, SKBR-3 and MCF-7) were transfected with siRNA against sortilin (siSORT) and universal negative control siRNA (siCONT). Scratching of the cell layer was performed 48 h after transfection and reduction in gap area was measured over 6 h. SiSORT inhibited migration only in MDA-MB-231 cells. B. Transwell invasion assay of MDA-MB-231 cells. Transwell assays were set up 48 h after siRNA transfection and cells were allowed to invade for 48 h. To take under account a potential impact of cell adhesion on the assay, cells were counted on both sides of the Transwell filter. Left panel, white columns represent the number of cells on the upper side of the filter, and the black columns the number of cells on the down side. Right panel, the percentage of invading cells in siSORT versus siCONT is represented. C. Western-blot detection of vimentin and activation of SRC, FAK, Akt and Erk1/2, 72 h after transfection with siSORT versus siCONT. Antibodies against vimentin, β-actin, SRC, phospho-SRC (Tyr416), FAK, phospho-FAK (Tyr576/577), Akt, phosphor-Akt (Ser473), Erk1/2, phosphor-Erk1/2 were used. For panel A and B, error bars represent SD. * p

    Article Snippet: The efficiency of sortilin knockdown was assessed by Western-blotting using anti-sortilin antibody (ANT009, Alomone Labs, Israel).

    Techniques: Migration, Wound Healing Assay, Multiple Displacement Amplification, Transfection, Negative Control, Transwell Invasion Assay, Western Blot, Activation Assay

    Immunohistological detection of sortilin in breast cancers The expression of sortilin was assessed by immunohistochemistry in a series of invasive breast cancers and normal adjacent tissues. Representative photos of sortilin immunolabeling are shown. A. Entire core and B, C. higher magnifications obtained for normal breast adjacent tissue; D. Entire core and E. ) higher magnification obtained for an invasive ductal carcinoma (IDC) positive for sortilin; F. Sortilin negative IDC. G. Entire core and H. higher magnification obtained for an invasive lobular carcinoma (ILC) positive for sortilin; I. Sortilin negative ILC. Magnification (20x, 200x) is indicated.

    Journal: Oncotarget

    Article Title: Sortilin is associated with breast cancer aggressiveness and contributes to tumor cell adhesion and invasion

    doi:

    Figure Lengend Snippet: Immunohistological detection of sortilin in breast cancers The expression of sortilin was assessed by immunohistochemistry in a series of invasive breast cancers and normal adjacent tissues. Representative photos of sortilin immunolabeling are shown. A. Entire core and B, C. higher magnifications obtained for normal breast adjacent tissue; D. Entire core and E. ) higher magnification obtained for an invasive ductal carcinoma (IDC) positive for sortilin; F. Sortilin negative IDC. G. Entire core and H. higher magnification obtained for an invasive lobular carcinoma (ILC) positive for sortilin; I. Sortilin negative ILC. Magnification (20x, 200x) is indicated.

    Article Snippet: The efficiency of sortilin knockdown was assessed by Western-blotting using anti-sortilin antibody (ANT009, Alomone Labs, Israel).

    Techniques: Expressing, Immunohistochemistry, Immunolabeling

    Expression of sortilin in breast cancer cell lines A. Quantitative RT-PCR analysis of sortilin gene expression in a range of breast cancer cell lines. Human mammary epithelial cells (HMEC) and the breast cancer cell lines MCF-7, MDA-MB-231 and their brain metastatic derivatives 231-BR, SKBR-3, MDA-MB-468, BT-474 and MDA-MB-453 were analyzed. Normalization was performed using β actin and the value obtained for HMEC was considered as 1. B. Western-blotting detection of sortilin in the same breast cancer cell lines. A band at about 100 kDa, the expected molecular weight of sortilin, was observed in all cell lines. In MCF-7, SKBR3 and BT474 cells, an additional band at 50 kDa was also detected. C. Sortilin was detected in the HMEC derivatives model of breast tumorigenic progression. The intensity of the sortilin band was higher in the tumorigenic HMLE and HMLER cells compared to the precancerous HME and the normal non-transformed HMEC.

    Journal: Oncotarget

    Article Title: Sortilin is associated with breast cancer aggressiveness and contributes to tumor cell adhesion and invasion

    doi:

    Figure Lengend Snippet: Expression of sortilin in breast cancer cell lines A. Quantitative RT-PCR analysis of sortilin gene expression in a range of breast cancer cell lines. Human mammary epithelial cells (HMEC) and the breast cancer cell lines MCF-7, MDA-MB-231 and their brain metastatic derivatives 231-BR, SKBR-3, MDA-MB-468, BT-474 and MDA-MB-453 were analyzed. Normalization was performed using β actin and the value obtained for HMEC was considered as 1. B. Western-blotting detection of sortilin in the same breast cancer cell lines. A band at about 100 kDa, the expected molecular weight of sortilin, was observed in all cell lines. In MCF-7, SKBR3 and BT474 cells, an additional band at 50 kDa was also detected. C. Sortilin was detected in the HMEC derivatives model of breast tumorigenic progression. The intensity of the sortilin band was higher in the tumorigenic HMLE and HMLER cells compared to the precancerous HME and the normal non-transformed HMEC.

    Article Snippet: The efficiency of sortilin knockdown was assessed by Western-blotting using anti-sortilin antibody (ANT009, Alomone Labs, Israel).

    Techniques: Expressing, Quantitative RT-PCR, Multiple Displacement Amplification, Western Blot, Molecular Weight, Transformation Assay

    Impact of sortilin knockdown on proliferation, survival and adhesion of breast cancer cells A. SiRNA against Sortilin (siSORT) and universal negative control siRNA (siCONT) were transfected in MDA-MB-231, MCF-7 and SKBR-3 breast cancer cells, and the impact on the level of sortilin was measured by Western-blotting 24, 48 and 72 h after transfection. Non-transfected cells (non transf.) were also analyzed. B. Microscopic observation of breast cancer cells 72 h after transfection with siSORT and siCONT. C. Counting of breast cancer cells 72 h after transfection with siSORT versus siCONT. The histograms represent the mean number of cells per well. D. Flow cytometry analysis of breast cancer cells 72 h after transfection with siSORT or siCONT. The percentage of cells in SG2M, G0/G1 and subG0/G1 is indicated. For each cell line, a picture of Hoechst staining observed in siSORT is shown. E. Impact of siRNA against sortilin on breast cancer cell adhesion. Breast cancer cells were transfected with siRNA and were seeded in culture dishes. 48 h latter, number of attached cells was counted at the indicated times after seeding. Results are expressed, as percentage of adherent cells. For panel C and E, error bars represent SD. * p

    Journal: Oncotarget

    Article Title: Sortilin is associated with breast cancer aggressiveness and contributes to tumor cell adhesion and invasion

    doi:

    Figure Lengend Snippet: Impact of sortilin knockdown on proliferation, survival and adhesion of breast cancer cells A. SiRNA against Sortilin (siSORT) and universal negative control siRNA (siCONT) were transfected in MDA-MB-231, MCF-7 and SKBR-3 breast cancer cells, and the impact on the level of sortilin was measured by Western-blotting 24, 48 and 72 h after transfection. Non-transfected cells (non transf.) were also analyzed. B. Microscopic observation of breast cancer cells 72 h after transfection with siSORT and siCONT. C. Counting of breast cancer cells 72 h after transfection with siSORT versus siCONT. The histograms represent the mean number of cells per well. D. Flow cytometry analysis of breast cancer cells 72 h after transfection with siSORT or siCONT. The percentage of cells in SG2M, G0/G1 and subG0/G1 is indicated. For each cell line, a picture of Hoechst staining observed in siSORT is shown. E. Impact of siRNA against sortilin on breast cancer cell adhesion. Breast cancer cells were transfected with siRNA and were seeded in culture dishes. 48 h latter, number of attached cells was counted at the indicated times after seeding. Results are expressed, as percentage of adherent cells. For panel C and E, error bars represent SD. * p

    Article Snippet: The efficiency of sortilin knockdown was assessed by Western-blotting using anti-sortilin antibody (ANT009, Alomone Labs, Israel).

    Techniques: Negative Control, Transfection, Multiple Displacement Amplification, Western Blot, Flow Cytometry, Cytometry, Staining

    Association of ARHGAP33 with schizophrenia. ( a , b ) Quantitative RT–PCR analysis of ARHGAP33 and SORT1 expression in immortalized lymphocytes from schizophrenia patients (schizo.) and age- and sex-matched controls. Significant reductions are observed in ARHGAP33 (each n =45, U =699, P =0.011, Mann–Whitney U -test; a ) and SORT1 (each n =45; U =683, P =7.8 × 10 −3 , Mann–Whitney U -test; b ) in schizophrenia patients. The expression levels of these genes were normalized to GAPDH mRNA. Bars show median values. ( c ) Linkage disequilibrium of ARHGAP33 in the HapMap JPT. Each diamond represents the correlation ( r 2 ) between each pair of SNPs, with darker shades representing stronger linkage disequilibrium, as obtained from the HapMap JST samples. ( d ) The locations of the SNPs analysed in this study. ( e , f ) Impact of the risk-T-allele on grey matter volume of the left middle temporal gyrus in schizophrenia patients. A significant cluster of the genotype effect in the left middle temporal gyrus is observed in schizophrenia patients, which is shown as cross-hairline (uncorrected P

    Journal: Nature Communications

    Article Title: Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders

    doi: 10.1038/ncomms10594

    Figure Lengend Snippet: Association of ARHGAP33 with schizophrenia. ( a , b ) Quantitative RT–PCR analysis of ARHGAP33 and SORT1 expression in immortalized lymphocytes from schizophrenia patients (schizo.) and age- and sex-matched controls. Significant reductions are observed in ARHGAP33 (each n =45, U =699, P =0.011, Mann–Whitney U -test; a ) and SORT1 (each n =45; U =683, P =7.8 × 10 −3 , Mann–Whitney U -test; b ) in schizophrenia patients. The expression levels of these genes were normalized to GAPDH mRNA. Bars show median values. ( c ) Linkage disequilibrium of ARHGAP33 in the HapMap JPT. Each diamond represents the correlation ( r 2 ) between each pair of SNPs, with darker shades representing stronger linkage disequilibrium, as obtained from the HapMap JST samples. ( d ) The locations of the SNPs analysed in this study. ( e , f ) Impact of the risk-T-allele on grey matter volume of the left middle temporal gyrus in schizophrenia patients. A significant cluster of the genotype effect in the left middle temporal gyrus is observed in schizophrenia patients, which is shown as cross-hairline (uncorrected P

    Article Snippet: Commercially available antibodies used were as follows: anti-ARHGAP33 (#HPA030118) antibody (Atlas Antibodies, Stockholm, Sweden), anti-GM130 (#610822) and anti-EEA1 (#9001964) antibodies (BD Transduction Laboratories, CA, USA), anti-PSD95 antibody (#MA1–046; Affinity Bioreagents, CO, USA), anti-TrkC (#3376) and anti-Tubulin (#2125) antibodies (Cell Signaling, MA, USA), anti-SORT1 (#ab16640) and anti-TrkB (#ab89925) antibodies (Abcam, Cambridge, UK), anti-SorLA (#sc136073) antibody (SantaCruz, CA, USA), anti-GM130 antibody (#G7295; Sigma, MO, USA), anti-phosphotyrosine (4G10) antibody (#05–1050; Millipore, MA, USA), anti-SORT1 antibody (#ANT-009; Alomone labs, Jerusalem, Israel), anti-Rab11 antibody (#71–5300; Invitrogen, MA, USA) and anti-Rab27 antibody (#18975; IBL, Gunma, Japan).

    Techniques: Quantitative RT-PCR, Expressing, MANN-WHITNEY

    Increased TrkB at the Golgi apparatus in ARHGAP33 KO mice. ( a ) ARHGAP33 was localized to the Golgi apparatus. Double immunostaining for ARHGAP33 and a Golgi marker, GM130, in dissociated hippocampal neurons. Scale bar, 5 μm. Asterisks indicate the nucleus of neurons. Note that ARHGAP33 immunoreactivity was not detected in the neurons from ARHGAP33 KO mice (lower). The data are representative of three independent experiments. ( b ) ARHGAP33 and SORT1 were co-fractionated with a Golgi marker, GM130. Biochemical preparation of the Golgi membrane fraction from a mouse brain lysate with a discontinuous sucrose density gradient. Equal amounts of protein were loaded into individual lanes and probed with antibodies against SORT1, ARHGAP33, GM130 (a Golgi marker) and EEA1 (an endosomal marker). ( c ) The amount of TrkB, but not SORT1, in the Golgi membrane-enriched fraction was significantly increased in ARHGAP33 KO mice. Equal amount of the Golgi membrane fractions from WT and ARHGAP33 KO mice were probed with anti-TrkB, anti-SORT1, anti-GM130 and anti-ARHGAP33 antibodies. ( d ) Quantification of the amount of TrkB and SORT1 in the Golgi membrane-enriched fraction (each, n =7, TrkB, P =0.0017; SORT1, P > 0.05, Mann–Whitney U -test). The levels of TrkB and SORT1 in the Golgi-enriched fraction from ARHGAP33 KO mice were normalized to those from WT mice (The averaged WT values were set to 100%). * P

    Journal: Nature Communications

    Article Title: Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders

    doi: 10.1038/ncomms10594

    Figure Lengend Snippet: Increased TrkB at the Golgi apparatus in ARHGAP33 KO mice. ( a ) ARHGAP33 was localized to the Golgi apparatus. Double immunostaining for ARHGAP33 and a Golgi marker, GM130, in dissociated hippocampal neurons. Scale bar, 5 μm. Asterisks indicate the nucleus of neurons. Note that ARHGAP33 immunoreactivity was not detected in the neurons from ARHGAP33 KO mice (lower). The data are representative of three independent experiments. ( b ) ARHGAP33 and SORT1 were co-fractionated with a Golgi marker, GM130. Biochemical preparation of the Golgi membrane fraction from a mouse brain lysate with a discontinuous sucrose density gradient. Equal amounts of protein were loaded into individual lanes and probed with antibodies against SORT1, ARHGAP33, GM130 (a Golgi marker) and EEA1 (an endosomal marker). ( c ) The amount of TrkB, but not SORT1, in the Golgi membrane-enriched fraction was significantly increased in ARHGAP33 KO mice. Equal amount of the Golgi membrane fractions from WT and ARHGAP33 KO mice were probed with anti-TrkB, anti-SORT1, anti-GM130 and anti-ARHGAP33 antibodies. ( d ) Quantification of the amount of TrkB and SORT1 in the Golgi membrane-enriched fraction (each, n =7, TrkB, P =0.0017; SORT1, P > 0.05, Mann–Whitney U -test). The levels of TrkB and SORT1 in the Golgi-enriched fraction from ARHGAP33 KO mice were normalized to those from WT mice (The averaged WT values were set to 100%). * P

    Article Snippet: Commercially available antibodies used were as follows: anti-ARHGAP33 (#HPA030118) antibody (Atlas Antibodies, Stockholm, Sweden), anti-GM130 (#610822) and anti-EEA1 (#9001964) antibodies (BD Transduction Laboratories, CA, USA), anti-PSD95 antibody (#MA1–046; Affinity Bioreagents, CO, USA), anti-TrkC (#3376) and anti-Tubulin (#2125) antibodies (Cell Signaling, MA, USA), anti-SORT1 (#ab16640) and anti-TrkB (#ab89925) antibodies (Abcam, Cambridge, UK), anti-SorLA (#sc136073) antibody (SantaCruz, CA, USA), anti-GM130 antibody (#G7295; Sigma, MO, USA), anti-phosphotyrosine (4G10) antibody (#05–1050; Millipore, MA, USA), anti-SORT1 antibody (#ANT-009; Alomone labs, Jerusalem, Israel), anti-Rab11 antibody (#71–5300; Invitrogen, MA, USA) and anti-Rab27 antibody (#18975; IBL, Gunma, Japan).

    Techniques: Mouse Assay, Double Immunostaining, Marker, MANN-WHITNEY

    ARHGAP33 promotes the interaction between TrkB and SORT1. ( a ) ARHGAP33 formed complexes with SORT1 and TrkB. ARHGAP33-immunoprecipitates (left) and hippocampal total lysates (right) were immunoblotted with the indicated antibodies. ( b – d ) ARHGAP33 formed complexes with SORT1 and TrkB. HEK293T cells were transfected with ARHGAP33, SORT1 and TrkB, as indicated. Immunoprecipitates were immunoblotted with the indicated antibodies. ( e ) Weakened interaction between TrkB and SORT1 in ARHGAP33 KO mice. SORT1 immunoprecipitates were immunoblotted with the indicated antibodies. Representative blots (left) and quantification of co-immunoprecipitated TrkB (right; each n =9; P =3.4 × 10 −4 , Mann–Whitney U -test). * P

    Journal: Nature Communications

    Article Title: Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders

    doi: 10.1038/ncomms10594

    Figure Lengend Snippet: ARHGAP33 promotes the interaction between TrkB and SORT1. ( a ) ARHGAP33 formed complexes with SORT1 and TrkB. ARHGAP33-immunoprecipitates (left) and hippocampal total lysates (right) were immunoblotted with the indicated antibodies. ( b – d ) ARHGAP33 formed complexes with SORT1 and TrkB. HEK293T cells were transfected with ARHGAP33, SORT1 and TrkB, as indicated. Immunoprecipitates were immunoblotted with the indicated antibodies. ( e ) Weakened interaction between TrkB and SORT1 in ARHGAP33 KO mice. SORT1 immunoprecipitates were immunoblotted with the indicated antibodies. Representative blots (left) and quantification of co-immunoprecipitated TrkB (right; each n =9; P =3.4 × 10 −4 , Mann–Whitney U -test). * P

    Article Snippet: Commercially available antibodies used were as follows: anti-ARHGAP33 (#HPA030118) antibody (Atlas Antibodies, Stockholm, Sweden), anti-GM130 (#610822) and anti-EEA1 (#9001964) antibodies (BD Transduction Laboratories, CA, USA), anti-PSD95 antibody (#MA1–046; Affinity Bioreagents, CO, USA), anti-TrkC (#3376) and anti-Tubulin (#2125) antibodies (Cell Signaling, MA, USA), anti-SORT1 (#ab16640) and anti-TrkB (#ab89925) antibodies (Abcam, Cambridge, UK), anti-SorLA (#sc136073) antibody (SantaCruz, CA, USA), anti-GM130 antibody (#G7295; Sigma, MO, USA), anti-phosphotyrosine (4G10) antibody (#05–1050; Millipore, MA, USA), anti-SORT1 antibody (#ANT-009; Alomone labs, Jerusalem, Israel), anti-Rab11 antibody (#71–5300; Invitrogen, MA, USA) and anti-Rab27 antibody (#18975; IBL, Gunma, Japan).

    Techniques: Transfection, Mouse Assay, Immunoprecipitation, MANN-WHITNEY

    Impaired TrkB trafficking to the cell surface at synapses in ARHGAP33 KO mice. ( a ) Protein structure of a brain-enriched SNX protein, ARHGAP33. ARHGAP33 has an N-terminal PX domain, an SH3 domain and a RhoGAP domain. ( b , c ) Decreased cell-surface expression of TrkB in ARHGAP33 KO mice. Biotinylated cell-surface proteins (upper) and total lysates (lower) of WT and ARHGAP33 KO neurons (14 DIV) were immunoblotted with anti-TrkB, anti-TrkC, anti-SORT1, anti-GAPDH and anti-ARHGAP33 antibodies. ( b ) Representative blots. ( c ) Quantification of surface expression (each, n =8; TrkB, P =7.8 × 10 −4 ; TrkC and SORT1, P > 0.05; Mann–Whitney U -test). The expression levels of TrkB, TrkC and SORT1 in ARHGAP33 KO neurons were normalized to those in WT neurons (The averaged WT values were set to 100%). ( d , e ) Decreased TrkB in the isolated PSD fraction of ARHGAP33 KO mice. The isolated PSD fraction and total lysates of WT and ARHGAP33 KO mice were immunoblotted with anti-TrkB, anti-PSD-95, anti-SORT1, and anti-ARHGAP33 antibodies. Representative blots ( d ). Quantification for the isolated PSD fraction (each, n =8, TrkB, P =7.7 × 10 −4 ; SORT1 and PSD-95, P > 0.05; Mann–Whitney U -test) and for the total lysate (each, n =8, P > 0.05; Mann–Whitney U -test; e ) The expression levels of TrkB, SORT1 and PSD-95 in the PSD fraction and total lysate from ARHGAP33 KO mice were normalized to those from WT mice (The averaged WT values were set to 100%). Note that the amounts of PSD-95 and SORT1 in the isolated PSD fraction from ARHGAP33 KO mice were not significantly different from those in the fraction from WT mice. * P

    Journal: Nature Communications

    Article Title: Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders

    doi: 10.1038/ncomms10594

    Figure Lengend Snippet: Impaired TrkB trafficking to the cell surface at synapses in ARHGAP33 KO mice. ( a ) Protein structure of a brain-enriched SNX protein, ARHGAP33. ARHGAP33 has an N-terminal PX domain, an SH3 domain and a RhoGAP domain. ( b , c ) Decreased cell-surface expression of TrkB in ARHGAP33 KO mice. Biotinylated cell-surface proteins (upper) and total lysates (lower) of WT and ARHGAP33 KO neurons (14 DIV) were immunoblotted with anti-TrkB, anti-TrkC, anti-SORT1, anti-GAPDH and anti-ARHGAP33 antibodies. ( b ) Representative blots. ( c ) Quantification of surface expression (each, n =8; TrkB, P =7.8 × 10 −4 ; TrkC and SORT1, P > 0.05; Mann–Whitney U -test). The expression levels of TrkB, TrkC and SORT1 in ARHGAP33 KO neurons were normalized to those in WT neurons (The averaged WT values were set to 100%). ( d , e ) Decreased TrkB in the isolated PSD fraction of ARHGAP33 KO mice. The isolated PSD fraction and total lysates of WT and ARHGAP33 KO mice were immunoblotted with anti-TrkB, anti-PSD-95, anti-SORT1, and anti-ARHGAP33 antibodies. Representative blots ( d ). Quantification for the isolated PSD fraction (each, n =8, TrkB, P =7.7 × 10 −4 ; SORT1 and PSD-95, P > 0.05; Mann–Whitney U -test) and for the total lysate (each, n =8, P > 0.05; Mann–Whitney U -test; e ) The expression levels of TrkB, SORT1 and PSD-95 in the PSD fraction and total lysate from ARHGAP33 KO mice were normalized to those from WT mice (The averaged WT values were set to 100%). Note that the amounts of PSD-95 and SORT1 in the isolated PSD fraction from ARHGAP33 KO mice were not significantly different from those in the fraction from WT mice. * P

    Article Snippet: Commercially available antibodies used were as follows: anti-ARHGAP33 (#HPA030118) antibody (Atlas Antibodies, Stockholm, Sweden), anti-GM130 (#610822) and anti-EEA1 (#9001964) antibodies (BD Transduction Laboratories, CA, USA), anti-PSD95 antibody (#MA1–046; Affinity Bioreagents, CO, USA), anti-TrkC (#3376) and anti-Tubulin (#2125) antibodies (Cell Signaling, MA, USA), anti-SORT1 (#ab16640) and anti-TrkB (#ab89925) antibodies (Abcam, Cambridge, UK), anti-SorLA (#sc136073) antibody (SantaCruz, CA, USA), anti-GM130 antibody (#G7295; Sigma, MO, USA), anti-phosphotyrosine (4G10) antibody (#05–1050; Millipore, MA, USA), anti-SORT1 antibody (#ANT-009; Alomone labs, Jerusalem, Israel), anti-Rab11 antibody (#71–5300; Invitrogen, MA, USA) and anti-Rab27 antibody (#18975; IBL, Gunma, Japan).

    Techniques: Mouse Assay, Expressing, MANN-WHITNEY, Isolation

    Cooperative facilitation of TrkB trafficking by ARHGAP33 and SORT1. ( a , b ) Increased TrkB surface expression by ARHGAP33 and SORT1. HEK293T cells were transfected with TrkB, ARHGAP33 and SORT1 as indicated in b (1–4). Representative data from eight independent experiments of immunostaining of surface TrkB in HEK293T cells with an antibody against the extracellular region of TrkB ( a ). Scale bar, 10 μm. Representative blots (left) and the quantification of the surface TrkB (right) ( b ). Biotinylated cell-surface proteins were immunoblotted with the indicated antibodies. The surface expression of TrkB was enhanced by simultaneous expression of ARHGAP33 and SORT1 compared with the expression of ARHGAP33 alone (each n =8; ARHGAP33 alone versus ARHGAP33 and SORT1, P =0.004, Kruskal–Wallis test followed by post hoc Steel–Dwass tests). Western blots show representative results from eight independent experiments. The averaged value of surface TrkB level in cells expressing ARHGAP33 alone (lane 3) was set to 100%. * P

    Journal: Nature Communications

    Article Title: Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders

    doi: 10.1038/ncomms10594

    Figure Lengend Snippet: Cooperative facilitation of TrkB trafficking by ARHGAP33 and SORT1. ( a , b ) Increased TrkB surface expression by ARHGAP33 and SORT1. HEK293T cells were transfected with TrkB, ARHGAP33 and SORT1 as indicated in b (1–4). Representative data from eight independent experiments of immunostaining of surface TrkB in HEK293T cells with an antibody against the extracellular region of TrkB ( a ). Scale bar, 10 μm. Representative blots (left) and the quantification of the surface TrkB (right) ( b ). Biotinylated cell-surface proteins were immunoblotted with the indicated antibodies. The surface expression of TrkB was enhanced by simultaneous expression of ARHGAP33 and SORT1 compared with the expression of ARHGAP33 alone (each n =8; ARHGAP33 alone versus ARHGAP33 and SORT1, P =0.004, Kruskal–Wallis test followed by post hoc Steel–Dwass tests). Western blots show representative results from eight independent experiments. The averaged value of surface TrkB level in cells expressing ARHGAP33 alone (lane 3) was set to 100%. * P

    Article Snippet: Commercially available antibodies used were as follows: anti-ARHGAP33 (#HPA030118) antibody (Atlas Antibodies, Stockholm, Sweden), anti-GM130 (#610822) and anti-EEA1 (#9001964) antibodies (BD Transduction Laboratories, CA, USA), anti-PSD95 antibody (#MA1–046; Affinity Bioreagents, CO, USA), anti-TrkC (#3376) and anti-Tubulin (#2125) antibodies (Cell Signaling, MA, USA), anti-SORT1 (#ab16640) and anti-TrkB (#ab89925) antibodies (Abcam, Cambridge, UK), anti-SorLA (#sc136073) antibody (SantaCruz, CA, USA), anti-GM130 antibody (#G7295; Sigma, MO, USA), anti-phosphotyrosine (4G10) antibody (#05–1050; Millipore, MA, USA), anti-SORT1 antibody (#ANT-009; Alomone labs, Jerusalem, Israel), anti-Rab11 antibody (#71–5300; Invitrogen, MA, USA) and anti-Rab27 antibody (#18975; IBL, Gunma, Japan).

    Techniques: Expressing, Transfection, Immunostaining, Western Blot

    Nerves in the tumor microenvironment of lung cancer do not express NGF, proNGF, TrkA, p75 NTR and sortilin. ( A ) Immunohistochemical detection of the pan-neuronal marker PGP9.5 was used to detect nerves in lung cancers. The expression of NGF ( B ), proNGF ( C ), TrkA ( D ), p75 NTR ( E ) and sortilin ( F ) was not detected in serial sections. Black arrows indicate a nerve trunk composed of many axons. Scale = 25 μm.

    Journal: Scientific Reports

    Article Title: The neurotrophic tyrosine kinase receptor TrkA and its ligand NGF are increased in squamous cell carcinomas of the lung

    doi: 10.1038/s41598-018-26408-2

    Figure Lengend Snippet: Nerves in the tumor microenvironment of lung cancer do not express NGF, proNGF, TrkA, p75 NTR and sortilin. ( A ) Immunohistochemical detection of the pan-neuronal marker PGP9.5 was used to detect nerves in lung cancers. The expression of NGF ( B ), proNGF ( C ), TrkA ( D ), p75 NTR ( E ) and sortilin ( F ) was not detected in serial sections. Black arrows indicate a nerve trunk composed of many axons. Scale = 25 μm.

    Article Snippet: The following primary antibodies were used at 1/500 dilution: anti-proNGF (#AB9040, Merck Millipore), anti-NGF (#ab52918, Abcam), anti-TrkA (#2508, Cell Signaling), anti-p75NTR (#4201, Cell Signaling), anti-sortilin (#ANT-009, Alomone Labs), anti-PGP9.5 (#ab15503, Abcam).

    Techniques: Immunohistochemistry, Marker, Expressing

    Model of sortilin regulation of transcription. Schematic diagram showing that sortilin has tumor suppressor-like activity, reducing co-oncogene transcription. EGF activates EGFR and induces its internalization as a homodimer or as a hetero dimer with sortilin. Osimertinib treatment promotes EGFR internalization and nuclear translocation. (1) Endocytosis of EGFR with sortilin can result in translocation of the complex into the nucleus, where it binds to chromatin at the TSS, thereby repressing RNA Pol II binding and cMyc co-oncogene transcription. (2) Excess EGFR homodimers imported into the nucleus bind to a specific chromatin area and trigger the recruitment of RNA Pol II, activating transcription.

    Journal: bioRxiv

    Article Title: Sortilin exhibits tumor suppressor-like activity by limiting EGFR transducing function

    doi: 10.1101/2021.05.12.443742

    Figure Lengend Snippet: Model of sortilin regulation of transcription. Schematic diagram showing that sortilin has tumor suppressor-like activity, reducing co-oncogene transcription. EGF activates EGFR and induces its internalization as a homodimer or as a hetero dimer with sortilin. Osimertinib treatment promotes EGFR internalization and nuclear translocation. (1) Endocytosis of EGFR with sortilin can result in translocation of the complex into the nucleus, where it binds to chromatin at the TSS, thereby repressing RNA Pol II binding and cMyc co-oncogene transcription. (2) Excess EGFR homodimers imported into the nucleus bind to a specific chromatin area and trigger the recruitment of RNA Pol II, activating transcription.

    Article Snippet: Immunoprecipitation assays were performed by mixing 50 μg DNA, 500 μL of 1X ChIP buffer with PIC, and 2 μg antibody to EGFR H11 (anti-EGFR H11, #MA5-13070, ThermoFisher Scientific™, France), sortilin (#ANT-009, Alomone, Israël), normal Rabbit IgG (#2729, Cell Signaling), or mouse (G3A1) mAb IgG1 isotype control (#5415S, Cell Signaling).

    Techniques: Activity Assay, Translocation Assay, Binding Assay

    Osimertinib enhances nuclear importation of EGFR. (a) EGFR localization was analyzed by confocal microscopy in A549 and H1975 cells, in the absence or presence of EGF stimulation (50 ng/mL for 30 min) or osimertinib treatment (1 μM for 24 h). Scale bar, 10 μm, yellow arrows show EGFR location. (b) Western blotting showing that treatment of A549 and H1975 cells with osimertinib (1 μM for 24 h) controlled EGFR and sortilin importation into isolated cell nuclei following cell fractionation. Molecular weight (MW) in kilo Daltons (kDa).

    Journal: bioRxiv

    Article Title: Sortilin exhibits tumor suppressor-like activity by limiting EGFR transducing function

    doi: 10.1101/2021.05.12.443742

    Figure Lengend Snippet: Osimertinib enhances nuclear importation of EGFR. (a) EGFR localization was analyzed by confocal microscopy in A549 and H1975 cells, in the absence or presence of EGF stimulation (50 ng/mL for 30 min) or osimertinib treatment (1 μM for 24 h). Scale bar, 10 μm, yellow arrows show EGFR location. (b) Western blotting showing that treatment of A549 and H1975 cells with osimertinib (1 μM for 24 h) controlled EGFR and sortilin importation into isolated cell nuclei following cell fractionation. Molecular weight (MW) in kilo Daltons (kDa).

    Article Snippet: Immunoprecipitation assays were performed by mixing 50 μg DNA, 500 μL of 1X ChIP buffer with PIC, and 2 μg antibody to EGFR H11 (anti-EGFR H11, #MA5-13070, ThermoFisher Scientific™, France), sortilin (#ANT-009, Alomone, Israël), normal Rabbit IgG (#2729, Cell Signaling), or mouse (G3A1) mAb IgG1 isotype control (#5415S, Cell Signaling).

    Techniques: Confocal Microscopy, Western Blot, Isolation, Cell Fractionation, Molecular Weight

    cMYC expression correlates inversely with SORT1 expression in vitro and in tumor samples. (a) Western blotting showing EGFR and sortilin expression in lysates of H1975 Tet-ON- SORT1 cells following incubation in the absence or presence of 100 nM doxycyclin (dox) for 24 h. (b) Anti-EGFR immunoprecipitation (IP) of isolated nuclei from H1975 Tet-ON- SORT1 cells following incubation in the absence or presence of 100 nM doxycyclin for 24 h and stimulation with 50 ng/mL EGF for 30 min and immunoblotting (IB) with anti-sortilin. (c) Comparison of CCND1 and cMYC mRNA levels in H1975 Tet-ON- SORT1 cells following incubation in the absence or presence of 100 nM doxycycline for 24 h. (d) Effects of doxycyclin on tumor induction by H1975 Tet-ON- SORT1 cells in NOD-SCID mice. H1975 Tet-ON- SORT1 cells were subcutaneously engrafted (3×10 6 cells/mouse) onto NOD-SCID mice. Fifteen days later, corresponding to the beginning of tumor development, mice were treated with 2 mg/mL doxycyclin in drinking water or drinking water alone, and tumor volumes were measured. Tumor growth curves are shown for mice treated with dox (orange curve) and for control mice (blue curve). (e) qPCR measurements of expression of CCND1 and cMYC mRNAs in tumors of mice treated with (blue bar) and without (orange bar) dox. (f-h) Measurements of SORT1 mRNA levels (Z-score) in normal and lung adenocarcinoma (ADC) tissue samples obtained from the (f) Limoges University Hospital cohort and data sets from references (g) 28 and (h) 29. (i) qPCR measurements of SORT1 mRNA levels in tumor samples from the Limoges University Hospital cohort at different stages. (j, k) Quantification of (j) CCND1 and (k) cMYC mRNA levels in tumor samples from the Limoges University Hospital cohort expressing the lowest and highest quartiles of sortilin expression. (l) Correlation between levels of cMYC and SORT1 mRNA levels in NSCLC patients in the TCGA database ( r =-0.24; p =8.7.10 -8 ) and (m) in solid cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) database ( r =-0.2; p =1.6.10 -8 ). Diagrams represent the correlation between SORT1 expression and cMYC expression. All values are expressed as means ± SD, ** p

    Journal: bioRxiv

    Article Title: Sortilin exhibits tumor suppressor-like activity by limiting EGFR transducing function

    doi: 10.1101/2021.05.12.443742

    Figure Lengend Snippet: cMYC expression correlates inversely with SORT1 expression in vitro and in tumor samples. (a) Western blotting showing EGFR and sortilin expression in lysates of H1975 Tet-ON- SORT1 cells following incubation in the absence or presence of 100 nM doxycyclin (dox) for 24 h. (b) Anti-EGFR immunoprecipitation (IP) of isolated nuclei from H1975 Tet-ON- SORT1 cells following incubation in the absence or presence of 100 nM doxycyclin for 24 h and stimulation with 50 ng/mL EGF for 30 min and immunoblotting (IB) with anti-sortilin. (c) Comparison of CCND1 and cMYC mRNA levels in H1975 Tet-ON- SORT1 cells following incubation in the absence or presence of 100 nM doxycycline for 24 h. (d) Effects of doxycyclin on tumor induction by H1975 Tet-ON- SORT1 cells in NOD-SCID mice. H1975 Tet-ON- SORT1 cells were subcutaneously engrafted (3×10 6 cells/mouse) onto NOD-SCID mice. Fifteen days later, corresponding to the beginning of tumor development, mice were treated with 2 mg/mL doxycyclin in drinking water or drinking water alone, and tumor volumes were measured. Tumor growth curves are shown for mice treated with dox (orange curve) and for control mice (blue curve). (e) qPCR measurements of expression of CCND1 and cMYC mRNAs in tumors of mice treated with (blue bar) and without (orange bar) dox. (f-h) Measurements of SORT1 mRNA levels (Z-score) in normal and lung adenocarcinoma (ADC) tissue samples obtained from the (f) Limoges University Hospital cohort and data sets from references (g) 28 and (h) 29. (i) qPCR measurements of SORT1 mRNA levels in tumor samples from the Limoges University Hospital cohort at different stages. (j, k) Quantification of (j) CCND1 and (k) cMYC mRNA levels in tumor samples from the Limoges University Hospital cohort expressing the lowest and highest quartiles of sortilin expression. (l) Correlation between levels of cMYC and SORT1 mRNA levels in NSCLC patients in the TCGA database ( r =-0.24; p =8.7.10 -8 ) and (m) in solid cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) database ( r =-0.2; p =1.6.10 -8 ). Diagrams represent the correlation between SORT1 expression and cMYC expression. All values are expressed as means ± SD, ** p

    Article Snippet: Immunoprecipitation assays were performed by mixing 50 μg DNA, 500 μL of 1X ChIP buffer with PIC, and 2 μg antibody to EGFR H11 (anti-EGFR H11, #MA5-13070, ThermoFisher Scientific™, France), sortilin (#ANT-009, Alomone, Israël), normal Rabbit IgG (#2729, Cell Signaling), or mouse (G3A1) mAb IgG1 isotype control (#5415S, Cell Signaling).

    Techniques: Expressing, In Vitro, Western Blot, Incubation, Immunoprecipitation, Isolation, Mouse Assay, Real-time Polymerase Chain Reaction

    Sortilin and EGFR interact together in the nuclei of cancer cells. (a) Proximity ligation assay (PLA) showing the interaction between sortilin and EGFR in the lung adenocarcinoma cell line A549 in the absence or presence of EGF (50 ng/mL) for 30 min. Red spots indicate sites of PLA amplification, reflecting interactions between sortilin and EGFR. Scale bar, 10 μm; white arrows show EGFR–sortilin clusters. ( b) Z-stack sections of confocal microscopy images showing sortilin and EGFR interactions in z axis (insets #2–26). White arrows show EGFR–sortilin clusters. (c) 3D confocal microscopy images showing EGFR–sortilin interactions at angles of 90° and 155°. (d) Quantification of EGFR–sortilin spots per nucleus, in the absence or presence of EGF for 5 or 30 min. (e) Estimated volumes of EGFR–sortilin clusters per nucleus (μm 3 /nucleus) in the absence or presence of EGF for 5 or 30 min. (f) Confirmation of EGFR–sortilin interactions by nuclear co-immunoprecipitation of A549 cell lysates in the absence or presence of EGF (50 ng/mL) for 30 min and immunoblotted (IB) with anti-EGFR antibodies. (g) Immunoblots showing kinetics of EGFR and sortilin nuclear importation following EGF stimulation of A549 cells. Nuclear fractions were obtained 0, 5, 15, and 30 min after stimulation with 50 ng/mL EGF. (h) EGFR silencing by specific siRNA transfection for 72 h before assessment of sortilin importation into the nucleus by western blotting. (i) Quantification of nuclear importation of EGFR and sortilin following EGF stimulation. Molecular weights (MW) are shown in kilo Daltons (kDa). (j) Relative optical density (ROD) of sortilin expression in isolated nuclei following EGFR depletion by siRNA. All values represent means ± SD. * p

    Journal: bioRxiv

    Article Title: Sortilin exhibits tumor suppressor-like activity by limiting EGFR transducing function

    doi: 10.1101/2021.05.12.443742

    Figure Lengend Snippet: Sortilin and EGFR interact together in the nuclei of cancer cells. (a) Proximity ligation assay (PLA) showing the interaction between sortilin and EGFR in the lung adenocarcinoma cell line A549 in the absence or presence of EGF (50 ng/mL) for 30 min. Red spots indicate sites of PLA amplification, reflecting interactions between sortilin and EGFR. Scale bar, 10 μm; white arrows show EGFR–sortilin clusters. ( b) Z-stack sections of confocal microscopy images showing sortilin and EGFR interactions in z axis (insets #2–26). White arrows show EGFR–sortilin clusters. (c) 3D confocal microscopy images showing EGFR–sortilin interactions at angles of 90° and 155°. (d) Quantification of EGFR–sortilin spots per nucleus, in the absence or presence of EGF for 5 or 30 min. (e) Estimated volumes of EGFR–sortilin clusters per nucleus (μm 3 /nucleus) in the absence or presence of EGF for 5 or 30 min. (f) Confirmation of EGFR–sortilin interactions by nuclear co-immunoprecipitation of A549 cell lysates in the absence or presence of EGF (50 ng/mL) for 30 min and immunoblotted (IB) with anti-EGFR antibodies. (g) Immunoblots showing kinetics of EGFR and sortilin nuclear importation following EGF stimulation of A549 cells. Nuclear fractions were obtained 0, 5, 15, and 30 min after stimulation with 50 ng/mL EGF. (h) EGFR silencing by specific siRNA transfection for 72 h before assessment of sortilin importation into the nucleus by western blotting. (i) Quantification of nuclear importation of EGFR and sortilin following EGF stimulation. Molecular weights (MW) are shown in kilo Daltons (kDa). (j) Relative optical density (ROD) of sortilin expression in isolated nuclei following EGFR depletion by siRNA. All values represent means ± SD. * p

    Article Snippet: Immunoprecipitation assays were performed by mixing 50 μg DNA, 500 μL of 1X ChIP buffer with PIC, and 2 μg antibody to EGFR H11 (anti-EGFR H11, #MA5-13070, ThermoFisher Scientific™, France), sortilin (#ANT-009, Alomone, Israël), normal Rabbit IgG (#2729, Cell Signaling), or mouse (G3A1) mAb IgG1 isotype control (#5415S, Cell Signaling).

    Techniques: Proximity Ligation Assay, Amplification, Confocal Microscopy, Immunoprecipitation, Western Blot, Transfection, Expressing, Isolation

    Sortilin overexpression increases sortilin chromatin binding on cMYC and limits polymerase II recruitment. (a-b) EGFR and sortilin ChIP-qPCR were performed on H1975 control cells transfected with empty vector (EV) or on H1975 sortilin overexpressing cells ( OE-SORT1 ) in the absence or presence of EGF (50 ng/mL) for 30 min. CCND1 and cMYC promoters were amplified by qPCR. (c) Pol II ChIP-qPCR performed on EV and OE-SORT1 cells. (d) Levels of CCND1 and cMYC mRNAs in control (EV) and OE-SORT1 cells by qPCR. All values represent means ± SD, * p

    Journal: bioRxiv

    Article Title: Sortilin exhibits tumor suppressor-like activity by limiting EGFR transducing function

    doi: 10.1101/2021.05.12.443742

    Figure Lengend Snippet: Sortilin overexpression increases sortilin chromatin binding on cMYC and limits polymerase II recruitment. (a-b) EGFR and sortilin ChIP-qPCR were performed on H1975 control cells transfected with empty vector (EV) or on H1975 sortilin overexpressing cells ( OE-SORT1 ) in the absence or presence of EGF (50 ng/mL) for 30 min. CCND1 and cMYC promoters were amplified by qPCR. (c) Pol II ChIP-qPCR performed on EV and OE-SORT1 cells. (d) Levels of CCND1 and cMYC mRNAs in control (EV) and OE-SORT1 cells by qPCR. All values represent means ± SD, * p

    Article Snippet: Immunoprecipitation assays were performed by mixing 50 μg DNA, 500 μL of 1X ChIP buffer with PIC, and 2 μg antibody to EGFR H11 (anti-EGFR H11, #MA5-13070, ThermoFisher Scientific™, France), sortilin (#ANT-009, Alomone, Israël), normal Rabbit IgG (#2729, Cell Signaling), or mouse (G3A1) mAb IgG1 isotype control (#5415S, Cell Signaling).

    Techniques: Over Expression, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Amplification

    Osimertinib increases EGFR and sortilin binding to chromatin. (a-d) Results of EGFR and sortilin ChIP-qPCR of A549 and H1975 cells incubated in the absence or presence of EGF (50 ng/mL for 30 min) or osimertinib (1 μM for 24 h). CCND1 and cMYC promoter sequences were amplified by qPCR. (e-f) Levels of CCND1 and cMYC mRNAs determined by RT-qPCR in A549 and H1975 cells in the absence or presence of osimertinib. (g) CCND1 and cMYC mRNAs were quantified by RT-qPCR in control H1975 cells carrying empty vector (EV) and H1975 cells overexpressing (OE-SORT1) in the presence of osimertinib. All values represent means ± SD, *** p

    Journal: bioRxiv

    Article Title: Sortilin exhibits tumor suppressor-like activity by limiting EGFR transducing function

    doi: 10.1101/2021.05.12.443742

    Figure Lengend Snippet: Osimertinib increases EGFR and sortilin binding to chromatin. (a-d) Results of EGFR and sortilin ChIP-qPCR of A549 and H1975 cells incubated in the absence or presence of EGF (50 ng/mL for 30 min) or osimertinib (1 μM for 24 h). CCND1 and cMYC promoter sequences were amplified by qPCR. (e-f) Levels of CCND1 and cMYC mRNAs determined by RT-qPCR in A549 and H1975 cells in the absence or presence of osimertinib. (g) CCND1 and cMYC mRNAs were quantified by RT-qPCR in control H1975 cells carrying empty vector (EV) and H1975 cells overexpressing (OE-SORT1) in the presence of osimertinib. All values represent means ± SD, *** p

    Article Snippet: Immunoprecipitation assays were performed by mixing 50 μg DNA, 500 μL of 1X ChIP buffer with PIC, and 2 μg antibody to EGFR H11 (anti-EGFR H11, #MA5-13070, ThermoFisher Scientific™, France), sortilin (#ANT-009, Alomone, Israël), normal Rabbit IgG (#2729, Cell Signaling), or mouse (G3A1) mAb IgG1 isotype control (#5415S, Cell Signaling).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Incubation, Amplification, Quantitative RT-PCR, Plasmid Preparation

    EGF stimulation increases EGFR and sortilin binding to chromatin. (a-b) Quantitative PCR (qPCR) of chromatin immunoprecipitated (ChIP) by anti-EGFR (blue bars) and anti-sortilin (orange bars) antibodies in control cells (pLKO) and cells depleted of EGFR or SORT1 mRNA by incubation with shRNAs, and incubated in the absence or presence of EGF (50 ng/mL) for 30 min. Histograms represented the percentages of input following normalization. CCND1 and cMYC promoters were amplified by qPCR. (c-d) RT-qPCR measurements of CCND1 and cMYC mRNAs in A549 cells depleted of EGFR or SORT1 mRNA with shRNAs and in control cells (pLKO). All values represent means ± SD, *** p

    Journal: bioRxiv

    Article Title: Sortilin exhibits tumor suppressor-like activity by limiting EGFR transducing function

    doi: 10.1101/2021.05.12.443742

    Figure Lengend Snippet: EGF stimulation increases EGFR and sortilin binding to chromatin. (a-b) Quantitative PCR (qPCR) of chromatin immunoprecipitated (ChIP) by anti-EGFR (blue bars) and anti-sortilin (orange bars) antibodies in control cells (pLKO) and cells depleted of EGFR or SORT1 mRNA by incubation with shRNAs, and incubated in the absence or presence of EGF (50 ng/mL) for 30 min. Histograms represented the percentages of input following normalization. CCND1 and cMYC promoters were amplified by qPCR. (c-d) RT-qPCR measurements of CCND1 and cMYC mRNAs in A549 cells depleted of EGFR or SORT1 mRNA with shRNAs and in control cells (pLKO). All values represent means ± SD, *** p

    Article Snippet: Immunoprecipitation assays were performed by mixing 50 μg DNA, 500 μL of 1X ChIP buffer with PIC, and 2 μg antibody to EGFR H11 (anti-EGFR H11, #MA5-13070, ThermoFisher Scientific™, France), sortilin (#ANT-009, Alomone, Israël), normal Rabbit IgG (#2729, Cell Signaling), or mouse (G3A1) mAb IgG1 isotype control (#5415S, Cell Signaling).

    Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, Chromatin Immunoprecipitation, Incubation, Amplification, Quantitative RT-PCR

    EGFR and sortilin interact with chromatin. (a) PCR amplification of CCND1 and cMYC promoter sequences in A549 cells stimulated with EGF (50 ng/mL) for 30 min following chromatin immunoprecipitation (ChIP) with either anti-EGFR or anti-sortilin antibodies. Respective isotype IgGs, IgG1 ChIP(EGFR) and IgG ChIP (sortilin), were used as controls and compared with input samples (input chromatin) corresponding to non-ChIP DNA as internal control. (b) Peaks enriched for EGFR and sortilin in A549 cells stimulated with EGF (50 ng/mL) for 30 min. (c) Distribution of EGFR and sortilin ChIP-Seq reads near 5 kb upstream /downstream of TSS. (d) ChiP-Seq overview shown with the IGV genome browser representing EGFR and sortilin binding sites on CCND1 and cMYC TSS. (e) Table showing Pearson correlation coefficients between each pair of ChIP conditions. Color intensity was representative of the magnitude of the correlation coefficient. (f) Common consensus sequences of EGFR and sortilin binding sites on chromatin.

    Journal: bioRxiv

    Article Title: Sortilin exhibits tumor suppressor-like activity by limiting EGFR transducing function

    doi: 10.1101/2021.05.12.443742

    Figure Lengend Snippet: EGFR and sortilin interact with chromatin. (a) PCR amplification of CCND1 and cMYC promoter sequences in A549 cells stimulated with EGF (50 ng/mL) for 30 min following chromatin immunoprecipitation (ChIP) with either anti-EGFR or anti-sortilin antibodies. Respective isotype IgGs, IgG1 ChIP(EGFR) and IgG ChIP (sortilin), were used as controls and compared with input samples (input chromatin) corresponding to non-ChIP DNA as internal control. (b) Peaks enriched for EGFR and sortilin in A549 cells stimulated with EGF (50 ng/mL) for 30 min. (c) Distribution of EGFR and sortilin ChIP-Seq reads near 5 kb upstream /downstream of TSS. (d) ChiP-Seq overview shown with the IGV genome browser representing EGFR and sortilin binding sites on CCND1 and cMYC TSS. (e) Table showing Pearson correlation coefficients between each pair of ChIP conditions. Color intensity was representative of the magnitude of the correlation coefficient. (f) Common consensus sequences of EGFR and sortilin binding sites on chromatin.

    Article Snippet: Immunoprecipitation assays were performed by mixing 50 μg DNA, 500 μL of 1X ChIP buffer with PIC, and 2 μg antibody to EGFR H11 (anti-EGFR H11, #MA5-13070, ThermoFisher Scientific™, France), sortilin (#ANT-009, Alomone, Israël), normal Rabbit IgG (#2729, Cell Signaling), or mouse (G3A1) mAb IgG1 isotype control (#5415S, Cell Signaling).

    Techniques: Polymerase Chain Reaction, Amplification, Chromatin Immunoprecipitation, Binding Assay