Structured Review

Santa Cruz Biotechnology pbst
Pbst, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1283 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pbst/product/Santa Cruz Biotechnology
Average 94 stars, based on 1283 article reviews
Price from $9.99 to $1999.99
pbst - by Bioz Stars, 2020-07
94/100 stars

Images

Related Articles

Incubation:

Article Title: Elevated IL-4 and IFN-γ Levels in Muscle Tissue of Patients with Dermatomyositis
Article Snippet: .. The blots were washed with PBST and incubated for 1 hour with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) in PBST. .. Immunoreactivity of the protein bands were detected by enhanced chemiluminescent autoradiography (ECL kit;Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Article Title: Temporal inhibition of mouse mammary gland cancer metastasis by CORM-A1 and DETA/NO combination therapy
Article Snippet: .. The membranes from the group A were washed three times for 10 min with PBST after reading, and then incubated for 1 h (RT) with an anti-β-actin-HRP monoclonal antibody (1:1,000; Santa Cruz Biot., Dallas, USA; group A). .. The membranes from the group B were washed three times for 5 min with TBST followed by 1 h (RT) incubation with mouse monoclonal anti-β-actin antibody (1:5,000; Cell Signaling Technology, Warsaw, Poland).

Article Title: (-)-Epigallocatechin-3-Gallate Ameliorates Learning and Memory Deficits by Adjusting the Balance of TrkA/p75NTR Signaling in APP/PS1 Transgenic Mice
Article Snippet: .. After washed with PBST, the membranes were incubated with goat horseradish peroxide-conjugated second rabbit anti-body (1:2,000; Santa Cruz) for 1 h at room temperature. .. Immunoreactive bands were visualized using the enhanced chemiluminescent kit (ECL+, Amersham Biosciences).

Article Title: miR-338-3p inhibits A549 lung cancer cell proliferation and invasion by targeting AKT and β-catenin signaling pathways
Article Snippet: .. Membranes were washed with PBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:200; cat. no. sc-2357; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 2 h at room temperature and washed with PBST. .. A chemiluminescent detection (Thermo Fisher Scientific, Inc.) of HRP activity was used to detect the signal in the membranes, and images were taken with chemiluminescence apparatus (Quantity One® software; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Article Title: Fulvestrant-Induced Cell Death and Proteasomal Degradation of Estrogen Receptor α Protein in MCF-7 Cells Require the CSK c-Src Tyrosine Kinase
Article Snippet: .. The membranes were washed with PBST and then incubated with peroxidase-conjugated secondary antibodies (donkey anti-goat IgG or goat anti-rabbit IgG, x3000 dilution, Santa Cruz Biotechnology) for 1 h at room temperature. ..

Article Title: PPARγ agonists regulate the expression of stemness and differentiation genes in brain tumour stem cells
Article Snippet: .. The residual binding sites in the membrane were blocked by incubating with PBST (PBS and 0.1% Tween 20) containing 5% non-fat dry milk powder for 1 h. The blots were incubated with rabbit anti-Sox2, rabbit anti-Nanog, goat anti-glial fibrillary acidic protein (GFAP), rabbit anti-chondroitin sulphate proteoglycan 4 (NG2), mouse anti-β -III Tubulin, mouse anti-CD133, or mouse anti-β -actin antibody (1 : 500, Santa Cruz Biotechnology, Santa Cruz, CA, USA) in PBST containing 1% non-fat milk powder at room temperature for 1 h. Membranes were then washed in PBST and incubated with corresponding peroxidase-conjugated anti-IgG antibody (1 : 2500, Santa Cruz Biotechnology) in PBS containing 1% milk powder for 1 h. The blots were then developed using Super-signal West-pico chemiluminescence reagent (Thermo Scientific, Rockford, IL, USA). .. Immunostaining and flow cytometry The effect of PPARγ agonists on the expansion of CD133+ , Sox2+ or Nanog+ BTSCs was analysed by immunostaining and flow cytometry.

Binding Assay:

Article Title: PPARγ agonists regulate the expression of stemness and differentiation genes in brain tumour stem cells
Article Snippet: .. The residual binding sites in the membrane were blocked by incubating with PBST (PBS and 0.1% Tween 20) containing 5% non-fat dry milk powder for 1 h. The blots were incubated with rabbit anti-Sox2, rabbit anti-Nanog, goat anti-glial fibrillary acidic protein (GFAP), rabbit anti-chondroitin sulphate proteoglycan 4 (NG2), mouse anti-β -III Tubulin, mouse anti-CD133, or mouse anti-β -actin antibody (1 : 500, Santa Cruz Biotechnology, Santa Cruz, CA, USA) in PBST containing 1% non-fat milk powder at room temperature for 1 h. Membranes were then washed in PBST and incubated with corresponding peroxidase-conjugated anti-IgG antibody (1 : 2500, Santa Cruz Biotechnology) in PBS containing 1% milk powder for 1 h. The blots were then developed using Super-signal West-pico chemiluminescence reagent (Thermo Scientific, Rockford, IL, USA). .. Immunostaining and flow cytometry The effect of PPARγ agonists on the expansion of CD133+ , Sox2+ or Nanog+ BTSCs was analysed by immunostaining and flow cytometry.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 84
    Santa Cruz Biotechnology pbst anti minpp1
    <t>MINPP1</t> absence impairs early neuronal differentiation. (A) Control (Ctrl-D10 and Ctrl-I004), patient-derived (CerID-30-2) and MINPP1 -/- iPSCs were differentiated towards neuronal lineage for 14 days. Representative images of the differentiated cells stained with early neuronal marker TUJ1 and neural progenitor marker PAX6. Hoechst was used as a nuclear stain. All scale bars correspond to 50μm. (B) Quantitative analysis of the immunofluorescence data. (Duplicate analysis of two independent experiments, one-way ANOVA, Tukey’s post hoc test, * and ** indicate p value: p
    Pbst Anti Minpp1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst anti minpp1/product/Santa Cruz Biotechnology
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pbst anti minpp1 - by Bioz Stars, 2020-07
    84/100 stars
      Buy from Supplier

    94
    Santa Cruz Biotechnology pbst
    <t>MINPP1</t> absence impairs early neuronal differentiation. (A) Control (Ctrl-D10 and Ctrl-I004), patient-derived (CerID-30-2) and MINPP1 -/- iPSCs were differentiated towards neuronal lineage for 14 days. Representative images of the differentiated cells stained with early neuronal marker TUJ1 and neural progenitor marker PAX6. Hoechst was used as a nuclear stain. All scale bars correspond to 50μm. (B) Quantitative analysis of the immunofluorescence data. (Duplicate analysis of two independent experiments, one-way ANOVA, Tukey’s post hoc test, * and ** indicate p value: p
    Pbst, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1283 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/Santa Cruz Biotechnology
    Average 94 stars, based on 1283 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

    92
    Santa Cruz Biotechnology triton x phosphate buffered saline pbst
    <t>MINPP1</t> absence impairs early neuronal differentiation. (A) Control (Ctrl-D10 and Ctrl-I004), patient-derived (CerID-30-2) and MINPP1 -/- iPSCs were differentiated towards neuronal lineage for 14 days. Representative images of the differentiated cells stained with early neuronal marker TUJ1 and neural progenitor marker PAX6. Hoechst was used as a nuclear stain. All scale bars correspond to 50μm. (B) Quantitative analysis of the immunofluorescence data. (Duplicate analysis of two independent experiments, one-way ANOVA, Tukey’s post hoc test, * and ** indicate p value: p
    Triton X Phosphate Buffered Saline Pbst, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/triton x phosphate buffered saline pbst/product/Santa Cruz Biotechnology
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    triton x phosphate buffered saline pbst - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    Image Search Results


    MINPP1 absence impairs early neuronal differentiation. (A) Control (Ctrl-D10 and Ctrl-I004), patient-derived (CerID-30-2) and MINPP1 -/- iPSCs were differentiated towards neuronal lineage for 14 days. Representative images of the differentiated cells stained with early neuronal marker TUJ1 and neural progenitor marker PAX6. Hoechst was used as a nuclear stain. All scale bars correspond to 50μm. (B) Quantitative analysis of the immunofluorescence data. (Duplicate analysis of two independent experiments, one-way ANOVA, Tukey’s post hoc test, * and ** indicate p value: p

    Journal: bioRxiv

    Article Title: MINPP1 prevents intracellular accumulation of the cation chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia

    doi: 10.1101/2020.05.17.100248

    Figure Lengend Snippet: MINPP1 absence impairs early neuronal differentiation. (A) Control (Ctrl-D10 and Ctrl-I004), patient-derived (CerID-30-2) and MINPP1 -/- iPSCs were differentiated towards neuronal lineage for 14 days. Representative images of the differentiated cells stained with early neuronal marker TUJ1 and neural progenitor marker PAX6. Hoechst was used as a nuclear stain. All scale bars correspond to 50μm. (B) Quantitative analysis of the immunofluorescence data. (Duplicate analysis of two independent experiments, one-way ANOVA, Tukey’s post hoc test, * and ** indicate p value: p

    Article Snippet: Nitrocellulose membranes were blocked either in Odyssey-TM Blocking Buffer (927-50003, LICOR) or in 5% dry milk diluted in 0.2% PBST for 1 hour and further incubated overnight at 4 °C with the following primary antibodies diluted in either OdysseyTM Blocking Buffer or 2.5% dry milk diluted in 0.2% PBST: Anti-MINPP1 (1:2000, sc-10399, Santa-Cruz), Anti-β Actin (1:5000, AM4302, Invitrogen), Anti-Calmodulin (1:1000, 465, Swant).

    Techniques: Derivative Assay, Staining, Marker, Immunofluorescence

    Phytase activity and SAX-HPLC analysis of extracellular and intracellular inositol phosphate levels. ( A )MINPP1 phytase activity in conditioned medium of MINPP1 -/- HEK293 cells upon addition of 4 nmol of IP 6 and incubation at 37°C for the time period indicated above. The samples were then mixed with Orange G (OrG) loading dye and resolved by PAGE followed by toluidine blue staining. Gel image representative of two independent experiments. Polyphosphate (PolyP) is shown as size ladder. ( B ) SAX-HPLC analysis of inositol phosphate levels in cell culture media of [ 3 H]-inositol labeled control and MINPP1 -/- HEK293 cells. (C-D) SAX-HPLC analysis of IP 2 and IP 6 levels in control and MINPP1 -/- HEK293 cells transiently transfected with plasmids encoding empty vector, wildtype, Y53D or E486K variant MINPP1. The data is representative of two independent experiments. [ 3 H]-IP n levels are presented as percentage of total radioactivity in the inositol-lipid fraction ([ 3 H]-PIP n ). Abbreviations used: IPn, inositol phosphates; PIPn, phosphatidyl inositol phosphates.

    Journal: bioRxiv

    Article Title: MINPP1 prevents intracellular accumulation of the cation chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia

    doi: 10.1101/2020.05.17.100248

    Figure Lengend Snippet: Phytase activity and SAX-HPLC analysis of extracellular and intracellular inositol phosphate levels. ( A )MINPP1 phytase activity in conditioned medium of MINPP1 -/- HEK293 cells upon addition of 4 nmol of IP 6 and incubation at 37°C for the time period indicated above. The samples were then mixed with Orange G (OrG) loading dye and resolved by PAGE followed by toluidine blue staining. Gel image representative of two independent experiments. Polyphosphate (PolyP) is shown as size ladder. ( B ) SAX-HPLC analysis of inositol phosphate levels in cell culture media of [ 3 H]-inositol labeled control and MINPP1 -/- HEK293 cells. (C-D) SAX-HPLC analysis of IP 2 and IP 6 levels in control and MINPP1 -/- HEK293 cells transiently transfected with plasmids encoding empty vector, wildtype, Y53D or E486K variant MINPP1. The data is representative of two independent experiments. [ 3 H]-IP n levels are presented as percentage of total radioactivity in the inositol-lipid fraction ([ 3 H]-PIP n ). Abbreviations used: IPn, inositol phosphates; PIPn, phosphatidyl inositol phosphates.

    Article Snippet: Nitrocellulose membranes were blocked either in Odyssey-TM Blocking Buffer (927-50003, LICOR) or in 5% dry milk diluted in 0.2% PBST for 1 hour and further incubated overnight at 4 °C with the following primary antibodies diluted in either OdysseyTM Blocking Buffer or 2.5% dry milk diluted in 0.2% PBST: Anti-MINPP1 (1:2000, sc-10399, Santa-Cruz), Anti-β Actin (1:5000, AM4302, Invitrogen), Anti-Calmodulin (1:1000, 465, Swant).

    Techniques: Activity Assay, High Performance Liquid Chromatography, Incubation, Polyacrylamide Gel Electrophoresis, Staining, Cell Culture, Labeling, Transfection, Plasmid Preparation, Variant Assay, Radioactivity

    Biallelic mutations in MINPP1 cause a distinct PCH phenotype. (A) Midline sagittal (top), coronal (middle) and axial (bottom) brain MRIs of control and patients from families CerID-30, CerID-11, CerID-09 and TR-PCH-01 respectively. Only sagittal (top) and coronal (middle) brain MRIs were available for the patient from the family PCH-2712 and sagittal brain MRI for the patients from PCH-2456 (top and middle). Sagittal MRIs show variable degree of brain stem (arrow) and cerebellar atrophy/hypoplasia (arrowhead). ( B) Schematic representation of the MINPP1 transcripts: NM_004897.5, NM_001178117.1 and NM_001178118.1 respectively. Exon numbers for the longest isoform NM_004897.5 are indicated above the schematic representation. Mutations are shown relative to their cDNA (NM_004897.5) position. ( C) Multiple-sequence alignment of MINPP1 from different species. Variant amino-acid residues p.Y53, p.F228, p.R401 and p.E486 are evolutionarily conserved. ( D) Linear schematic representation of MINPP1, showing the position of mutations with respect to predicted protein domains. Abbreviations used: Endoplasmic reticulum (ER).

    Journal: bioRxiv

    Article Title: MINPP1 prevents intracellular accumulation of the cation chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia

    doi: 10.1101/2020.05.17.100248

    Figure Lengend Snippet: Biallelic mutations in MINPP1 cause a distinct PCH phenotype. (A) Midline sagittal (top), coronal (middle) and axial (bottom) brain MRIs of control and patients from families CerID-30, CerID-11, CerID-09 and TR-PCH-01 respectively. Only sagittal (top) and coronal (middle) brain MRIs were available for the patient from the family PCH-2712 and sagittal brain MRI for the patients from PCH-2456 (top and middle). Sagittal MRIs show variable degree of brain stem (arrow) and cerebellar atrophy/hypoplasia (arrowhead). ( B) Schematic representation of the MINPP1 transcripts: NM_004897.5, NM_001178117.1 and NM_001178118.1 respectively. Exon numbers for the longest isoform NM_004897.5 are indicated above the schematic representation. Mutations are shown relative to their cDNA (NM_004897.5) position. ( C) Multiple-sequence alignment of MINPP1 from different species. Variant amino-acid residues p.Y53, p.F228, p.R401 and p.E486 are evolutionarily conserved. ( D) Linear schematic representation of MINPP1, showing the position of mutations with respect to predicted protein domains. Abbreviations used: Endoplasmic reticulum (ER).

    Article Snippet: Nitrocellulose membranes were blocked either in Odyssey-TM Blocking Buffer (927-50003, LICOR) or in 5% dry milk diluted in 0.2% PBST for 1 hour and further incubated overnight at 4 °C with the following primary antibodies diluted in either OdysseyTM Blocking Buffer or 2.5% dry milk diluted in 0.2% PBST: Anti-MINPP1 (1:2000, sc-10399, Santa-Cruz), Anti-β Actin (1:5000, AM4302, Invitrogen), Anti-Calmodulin (1:1000, 465, Swant).

    Techniques: Magnetic Resonance Imaging, Sequencing, Variant Assay

    Characterization of patient-derived (CerID-30-2) and MINPP1 -/- iPSCs and their neural derivatives. (A) Immunofluorescence staining of embryonic stem cell markers OCT4 and SOX2 in control (Ctrl-D10 and Ctrl-I004), patient-derived (CerID-30-2), and MINPP1 -/- iPSCs. Hoechst was used as a nuclear stain. All scale bars correspond to 50μm. (B) Western blot data showing the absence of MINPP1 in patient-derived CerID-30-2 and MINPP1 -/- iPSCs. MINPP1 (48 kDa) is present in controls (D10 and I004) iPSC lines (lower band). β-actin is shown as loading control. (C) Flow cytometer analysis of P75 and HNK1 in control (Ctrl-D10 and Ctrl-I004), patient-derived (CerID-30-2) and MINPP1 -/- neural rosette cultures.

    Journal: bioRxiv

    Article Title: MINPP1 prevents intracellular accumulation of the cation chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia

    doi: 10.1101/2020.05.17.100248

    Figure Lengend Snippet: Characterization of patient-derived (CerID-30-2) and MINPP1 -/- iPSCs and their neural derivatives. (A) Immunofluorescence staining of embryonic stem cell markers OCT4 and SOX2 in control (Ctrl-D10 and Ctrl-I004), patient-derived (CerID-30-2), and MINPP1 -/- iPSCs. Hoechst was used as a nuclear stain. All scale bars correspond to 50μm. (B) Western blot data showing the absence of MINPP1 in patient-derived CerID-30-2 and MINPP1 -/- iPSCs. MINPP1 (48 kDa) is present in controls (D10 and I004) iPSC lines (lower band). β-actin is shown as loading control. (C) Flow cytometer analysis of P75 and HNK1 in control (Ctrl-D10 and Ctrl-I004), patient-derived (CerID-30-2) and MINPP1 -/- neural rosette cultures.

    Article Snippet: Nitrocellulose membranes were blocked either in Odyssey-TM Blocking Buffer (927-50003, LICOR) or in 5% dry milk diluted in 0.2% PBST for 1 hour and further incubated overnight at 4 °C with the following primary antibodies diluted in either OdysseyTM Blocking Buffer or 2.5% dry milk diluted in 0.2% PBST: Anti-MINPP1 (1:2000, sc-10399, Santa-Cruz), Anti-β Actin (1:5000, AM4302, Invitrogen), Anti-Calmodulin (1:1000, 465, Swant).

    Techniques: Derivative Assay, Immunofluorescence, Staining, Western Blot, Flow Cytometry

    MINPP1 absence leads to disruption in inositol phosphates metabolism. (A-F) SAX-HPLC analysis of inositol phosphate levels in MINPP1 -/- HEK293 cells (A) , patient fibroblasts (CerID-30-1 and CerID-30-2) (B) , patient-derived (CerID-30-2) (C) and MINPP1 -/- iPSCs (D) , and their day-10 differentiating neuron counterparts (E-F) . The peaks ([ 3 H]-IP n ) were identified based on comparison to standards. [ 3 H]-IP n levels are presented as percentage of total radioactivity in the inositol-lipid fraction ([ 3 H]-PIP n ). All error bars represent standard deviation (s.d.). (n=2, Two-tailed student’s t-test for HEK293 and fibroblast data; One-way ANOVA for iPSCs and their differentiated counterparts data, Tukey’s post hoc test, *, **, ***, **** indicate p values p

    Journal: bioRxiv

    Article Title: MINPP1 prevents intracellular accumulation of the cation chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia

    doi: 10.1101/2020.05.17.100248

    Figure Lengend Snippet: MINPP1 absence leads to disruption in inositol phosphates metabolism. (A-F) SAX-HPLC analysis of inositol phosphate levels in MINPP1 -/- HEK293 cells (A) , patient fibroblasts (CerID-30-1 and CerID-30-2) (B) , patient-derived (CerID-30-2) (C) and MINPP1 -/- iPSCs (D) , and their day-10 differentiating neuron counterparts (E-F) . The peaks ([ 3 H]-IP n ) were identified based on comparison to standards. [ 3 H]-IP n levels are presented as percentage of total radioactivity in the inositol-lipid fraction ([ 3 H]-PIP n ). All error bars represent standard deviation (s.d.). (n=2, Two-tailed student’s t-test for HEK293 and fibroblast data; One-way ANOVA for iPSCs and their differentiated counterparts data, Tukey’s post hoc test, *, **, ***, **** indicate p values p

    Article Snippet: Nitrocellulose membranes were blocked either in Odyssey-TM Blocking Buffer (927-50003, LICOR) or in 5% dry milk diluted in 0.2% PBST for 1 hour and further incubated overnight at 4 °C with the following primary antibodies diluted in either OdysseyTM Blocking Buffer or 2.5% dry milk diluted in 0.2% PBST: Anti-MINPP1 (1:2000, sc-10399, Santa-Cruz), Anti-β Actin (1:5000, AM4302, Invitrogen), Anti-Calmodulin (1:1000, 465, Swant).

    Techniques: High Performance Liquid Chromatography, Derivative Assay, Radioactivity, Standard Deviation, Two Tailed Test

    PCH-associated mutations of MINPP1 are deleterious for protein function. (A) Schematic representation of inositol phosphate cycle. Abbreviations used: myo- inositol (Inositol) phosphatidylinositol (PI); phosphatidylinositol phosphate (PIP); phosphatidylinositol 4,5-bisphosphate (PIP 2 ); diacylglycerol (DAG); Phospholipase C (PLC); inositol phosphate (IP); inositol bisphosphate (IP 2 ); inositol 1,4,5-trisphosphate (IP 3 ); inositol 1,3,4,5-tetrakisphosphate (IP 4 ); inositol pentakisphosphate (IP 5 ); inositol hexakisphosphate (IP 6 ); Inositol Polyphosphate Multikinase (IPMK); I(1,4,5)P 3 3-Kinase (IP 3 -3K); Inositol-Pentakisphosphate 2-Kinase (IPPK); Multiple Inositol-Polyphosphate Phosphatase 1 (MINPP1). ( B-C ) Western blot analysis of MINPP1 level in patient fibroblasts and HEK293 cells with β-Actin shown as loading control. Patient fibroblasts CerID-30-1 and CerID-30-2 ( B ) and MINPP1 -/- HEK293 clones ( C ) show absent MINPP1. ( D) Assessment of cell proliferation by MTT assay. For each clone, MTT absorbance was measured 3, 24 and 48 hours post-seeding. Values represent the mean ± s.d. of triplicate determinations from 4 replicates. (n=4, Two-tailed student’s t-test, ** and *** indicate the p values p

    Journal: bioRxiv

    Article Title: MINPP1 prevents intracellular accumulation of the cation chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia

    doi: 10.1101/2020.05.17.100248

    Figure Lengend Snippet: PCH-associated mutations of MINPP1 are deleterious for protein function. (A) Schematic representation of inositol phosphate cycle. Abbreviations used: myo- inositol (Inositol) phosphatidylinositol (PI); phosphatidylinositol phosphate (PIP); phosphatidylinositol 4,5-bisphosphate (PIP 2 ); diacylglycerol (DAG); Phospholipase C (PLC); inositol phosphate (IP); inositol bisphosphate (IP 2 ); inositol 1,4,5-trisphosphate (IP 3 ); inositol 1,3,4,5-tetrakisphosphate (IP 4 ); inositol pentakisphosphate (IP 5 ); inositol hexakisphosphate (IP 6 ); Inositol Polyphosphate Multikinase (IPMK); I(1,4,5)P 3 3-Kinase (IP 3 -3K); Inositol-Pentakisphosphate 2-Kinase (IPPK); Multiple Inositol-Polyphosphate Phosphatase 1 (MINPP1). ( B-C ) Western blot analysis of MINPP1 level in patient fibroblasts and HEK293 cells with β-Actin shown as loading control. Patient fibroblasts CerID-30-1 and CerID-30-2 ( B ) and MINPP1 -/- HEK293 clones ( C ) show absent MINPP1. ( D) Assessment of cell proliferation by MTT assay. For each clone, MTT absorbance was measured 3, 24 and 48 hours post-seeding. Values represent the mean ± s.d. of triplicate determinations from 4 replicates. (n=4, Two-tailed student’s t-test, ** and *** indicate the p values p

    Article Snippet: Nitrocellulose membranes were blocked either in Odyssey-TM Blocking Buffer (927-50003, LICOR) or in 5% dry milk diluted in 0.2% PBST for 1 hour and further incubated overnight at 4 °C with the following primary antibodies diluted in either OdysseyTM Blocking Buffer or 2.5% dry milk diluted in 0.2% PBST: Anti-MINPP1 (1:2000, sc-10399, Santa-Cruz), Anti-β Actin (1:5000, AM4302, Invitrogen), Anti-Calmodulin (1:1000, 465, Swant).

    Techniques: Planar Chromatography, Western Blot, Clone Assay, MTT Assay, Two Tailed Test

    Altered iron and calcium homeostasis in the absence of MINPP1 enzyme in HEK293 and Minpp1 -/- mouse neural progenitor cells. (A-D) Quantification of total iron content ( A ), free iron (Fe 2+ and Fe 3+ ) ( B ), Fe 3+ ( C ) and Fe 2+ ( D ) levels in extracts from control and MINPP1 -/- HEK293 cells grown under low (-FAC) and high iron (+FAC, 100 µM) conditions. All values are normalized to the total protein concentration and represent the mean ± s.d (n=3, Two way ANOVA Sidak test, **, *** indicate p value p

    Journal: bioRxiv

    Article Title: MINPP1 prevents intracellular accumulation of the cation chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia

    doi: 10.1101/2020.05.17.100248

    Figure Lengend Snippet: Altered iron and calcium homeostasis in the absence of MINPP1 enzyme in HEK293 and Minpp1 -/- mouse neural progenitor cells. (A-D) Quantification of total iron content ( A ), free iron (Fe 2+ and Fe 3+ ) ( B ), Fe 3+ ( C ) and Fe 2+ ( D ) levels in extracts from control and MINPP1 -/- HEK293 cells grown under low (-FAC) and high iron (+FAC, 100 µM) conditions. All values are normalized to the total protein concentration and represent the mean ± s.d (n=3, Two way ANOVA Sidak test, **, *** indicate p value p

    Article Snippet: Nitrocellulose membranes were blocked either in Odyssey-TM Blocking Buffer (927-50003, LICOR) or in 5% dry milk diluted in 0.2% PBST for 1 hour and further incubated overnight at 4 °C with the following primary antibodies diluted in either OdysseyTM Blocking Buffer or 2.5% dry milk diluted in 0.2% PBST: Anti-MINPP1 (1:2000, sc-10399, Santa-Cruz), Anti-β Actin (1:5000, AM4302, Invitrogen), Anti-Calmodulin (1:1000, 465, Swant).

    Techniques: Protein Concentration

    Generation and characterization of CRISPR-Cas9 Minpp1 -/- mouse and Ca 2+ assays on HEK293 cell lines. (A) Schema of the sgRNA targeting site and the location of PCR primers. Abbreviations used: Primer Forward (PF) and Primer Reverse (PR). ( B ) PCR based genotyping of Minpp1 -/- mice indicated successful knockout alleles showing a 31 bp deletion and a smaller PCR product. ( C ) Representative images of hematoxylin-eosin (HE) staining on P21 sagittal slices of control and mutant cerebellum, scale bar 1 mm. ( D ) Quantification of total brain weight in grams (g) from P21 and 11-month old WT and Minpp1 -/- mice. (N=6 mice, Two-tailed student’s t-test, ** indicates p value p

    Journal: bioRxiv

    Article Title: MINPP1 prevents intracellular accumulation of the cation chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia

    doi: 10.1101/2020.05.17.100248

    Figure Lengend Snippet: Generation and characterization of CRISPR-Cas9 Minpp1 -/- mouse and Ca 2+ assays on HEK293 cell lines. (A) Schema of the sgRNA targeting site and the location of PCR primers. Abbreviations used: Primer Forward (PF) and Primer Reverse (PR). ( B ) PCR based genotyping of Minpp1 -/- mice indicated successful knockout alleles showing a 31 bp deletion and a smaller PCR product. ( C ) Representative images of hematoxylin-eosin (HE) staining on P21 sagittal slices of control and mutant cerebellum, scale bar 1 mm. ( D ) Quantification of total brain weight in grams (g) from P21 and 11-month old WT and Minpp1 -/- mice. (N=6 mice, Two-tailed student’s t-test, ** indicates p value p

    Article Snippet: Nitrocellulose membranes were blocked either in Odyssey-TM Blocking Buffer (927-50003, LICOR) or in 5% dry milk diluted in 0.2% PBST for 1 hour and further incubated overnight at 4 °C with the following primary antibodies diluted in either OdysseyTM Blocking Buffer or 2.5% dry milk diluted in 0.2% PBST: Anti-MINPP1 (1:2000, sc-10399, Santa-Cruz), Anti-β Actin (1:5000, AM4302, Invitrogen), Anti-Calmodulin (1:1000, 465, Swant).

    Techniques: CRISPR, Polymerase Chain Reaction, Mouse Assay, Knock-Out, Staining, Mutagenesis, Two Tailed Test

    Expression profile of MINPP1 in different human tissues and protein levels of MINPP1 variants and their corresponding glycosylation status in HEK293 cells. (A) Quantitative PCR analysis of human MINPP1 expression, normalized with ACTB , in human cells and tissues. Abbreviations used: Neural Stem Cells (NSCs); Neuronal Progenitor Cells (NPCs) (B) Control and MINPP1 -/- HEK293 were cultured for 48 hours in IncuCyte live-cell analysis system and cell confluence (%) was analysed in IncuCyte Zoom live-cell-imaging software. Graph shows the percentage of cell confluence (± s.d.) over time. (C) Control and MINPP1 -/- HEK293 were cultured with Annexin-V containing growth media for 48 hours inside the IncuCyte live-cell analysis system, and images were captured with red fluorescence channel to detect Annexin-V positive cells. Graph shows the average ratio (±s.d.) of red cell object count per mm 2 (n=3, two-tailed student’s t-test, not significant) (D-E) Western blot data showing the exogenous MINPP1 levels (D) and its glycosylated and deglycosylated forms (E) in MINPP1 -/- HEK293 cells transiently transfected with plasmids encoding empty vector, wild type, Y53D or E486K t MINPP1. MINPP1 is present in control HEK293 cells and absent in MINPP1 -/- HEK293 cells. β-Actin shown as loading control.

    Journal: bioRxiv

    Article Title: MINPP1 prevents intracellular accumulation of the cation chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia

    doi: 10.1101/2020.05.17.100248

    Figure Lengend Snippet: Expression profile of MINPP1 in different human tissues and protein levels of MINPP1 variants and their corresponding glycosylation status in HEK293 cells. (A) Quantitative PCR analysis of human MINPP1 expression, normalized with ACTB , in human cells and tissues. Abbreviations used: Neural Stem Cells (NSCs); Neuronal Progenitor Cells (NPCs) (B) Control and MINPP1 -/- HEK293 were cultured for 48 hours in IncuCyte live-cell analysis system and cell confluence (%) was analysed in IncuCyte Zoom live-cell-imaging software. Graph shows the percentage of cell confluence (± s.d.) over time. (C) Control and MINPP1 -/- HEK293 were cultured with Annexin-V containing growth media for 48 hours inside the IncuCyte live-cell analysis system, and images were captured with red fluorescence channel to detect Annexin-V positive cells. Graph shows the average ratio (±s.d.) of red cell object count per mm 2 (n=3, two-tailed student’s t-test, not significant) (D-E) Western blot data showing the exogenous MINPP1 levels (D) and its glycosylated and deglycosylated forms (E) in MINPP1 -/- HEK293 cells transiently transfected with plasmids encoding empty vector, wild type, Y53D or E486K t MINPP1. MINPP1 is present in control HEK293 cells and absent in MINPP1 -/- HEK293 cells. β-Actin shown as loading control.

    Article Snippet: Nitrocellulose membranes were blocked either in Odyssey-TM Blocking Buffer (927-50003, LICOR) or in 5% dry milk diluted in 0.2% PBST for 1 hour and further incubated overnight at 4 °C with the following primary antibodies diluted in either OdysseyTM Blocking Buffer or 2.5% dry milk diluted in 0.2% PBST: Anti-MINPP1 (1:2000, sc-10399, Santa-Cruz), Anti-β Actin (1:5000, AM4302, Invitrogen), Anti-Calmodulin (1:1000, 465, Swant).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Live Cell Imaging, Software, Fluorescence, Two Tailed Test, Western Blot, Transfection, Plasmid Preparation