Structured Review

Santa Cruz Biotechnology filipin
HEPES-Induced Protein Transfection Was Accomplished through Endocytosis and Charge Neutralization (A–C) MDAH2774 cells were pretreated for 30 min with Opti-MEM (Ctrl), 0.4 M sucrose (an inhibitor of clathrin-mediated endocytosis), 5 μg/mL <t>filipin</t> (an inhibitor of caveolae-mediated endocytosis), or 0.25 mM amiloride (an inhibitor of macropinocytosis). Alexa Fluor 488-conjugated antibodies were delivered into cells using 20 mM HEPES, as described in The HEPES Method , followed by a 4-h incubation period. (A) Transfection efficiency was determined using a confocal fluorescent microscope. (B) Alexa Fluor 488 fluorescent intensity signals were quantified using Q-Win software and are expressed as relative ratios. (C) At 4 h of inhibitor treatment, cytotoxicity was analyzed using the LDH assay. Error bars indicate the SEM (n = 3).*p
Filipin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Utilization of HEPES for Enhancing Protein Transfection into Mammalian Cells"

Article Title: Utilization of HEPES for Enhancing Protein Transfection into Mammalian Cells

Journal: Molecular Therapy. Methods & Clinical Development

doi: 10.1016/j.omtm.2018.12.005

HEPES-Induced Protein Transfection Was Accomplished through Endocytosis and Charge Neutralization (A–C) MDAH2774 cells were pretreated for 30 min with Opti-MEM (Ctrl), 0.4 M sucrose (an inhibitor of clathrin-mediated endocytosis), 5 μg/mL filipin (an inhibitor of caveolae-mediated endocytosis), or 0.25 mM amiloride (an inhibitor of macropinocytosis). Alexa Fluor 488-conjugated antibodies were delivered into cells using 20 mM HEPES, as described in The HEPES Method , followed by a 4-h incubation period. (A) Transfection efficiency was determined using a confocal fluorescent microscope. (B) Alexa Fluor 488 fluorescent intensity signals were quantified using Q-Win software and are expressed as relative ratios. (C) At 4 h of inhibitor treatment, cytotoxicity was analyzed using the LDH assay. Error bars indicate the SEM (n = 3).*p
Figure Legend Snippet: HEPES-Induced Protein Transfection Was Accomplished through Endocytosis and Charge Neutralization (A–C) MDAH2774 cells were pretreated for 30 min with Opti-MEM (Ctrl), 0.4 M sucrose (an inhibitor of clathrin-mediated endocytosis), 5 μg/mL filipin (an inhibitor of caveolae-mediated endocytosis), or 0.25 mM amiloride (an inhibitor of macropinocytosis). Alexa Fluor 488-conjugated antibodies were delivered into cells using 20 mM HEPES, as described in The HEPES Method , followed by a 4-h incubation period. (A) Transfection efficiency was determined using a confocal fluorescent microscope. (B) Alexa Fluor 488 fluorescent intensity signals were quantified using Q-Win software and are expressed as relative ratios. (C) At 4 h of inhibitor treatment, cytotoxicity was analyzed using the LDH assay. Error bars indicate the SEM (n = 3).*p

Techniques Used: Transfection, Neutralization, Incubation, Microscopy, Software, Lactate Dehydrogenase Assay

2) Product Images from "Inflammation-activated CXCL16 pathway contributes to tubulointerstitial injury in mouse diabetic nephropathy"

Article Title: Inflammation-activated CXCL16 pathway contributes to tubulointerstitial injury in mouse diabetic nephropathy

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2017.177

Inflammation increased lipid accumulation to accelerate tubulointerstitial injuries. Eight-week-old diabetic db/db mice were randomly assigned and received subcutaneous injections with either 0.5 mL distilled water ( db/db , n =10) or 0.5 mL 10% casein ( db/db +casein, n =10) every other day for 8 weeks. HK-2 cells were made quiescent using serum-free medium for 24 h, and treated with 30 mmol/L glucose (HG) or 30 mmol/L glucose plus 5 ng/mL IL-1β. TG, triglyceride; TC, total cholesterol; LDL, low-density lipoprotein; HDL, high-density lipoprotein (A). Filipin staining was used to examine lipid accumulation both in vivo and in vitro (B and D, original magnification ×200). The concentration of cholesterol ester was measured both in vivo (C, n =5) and in vitro (E). The results represent the mean±SD. * P
Figure Legend Snippet: Inflammation increased lipid accumulation to accelerate tubulointerstitial injuries. Eight-week-old diabetic db/db mice were randomly assigned and received subcutaneous injections with either 0.5 mL distilled water ( db/db , n =10) or 0.5 mL 10% casein ( db/db +casein, n =10) every other day for 8 weeks. HK-2 cells were made quiescent using serum-free medium for 24 h, and treated with 30 mmol/L glucose (HG) or 30 mmol/L glucose plus 5 ng/mL IL-1β. TG, triglyceride; TC, total cholesterol; LDL, low-density lipoprotein; HDL, high-density lipoprotein (A). Filipin staining was used to examine lipid accumulation both in vivo and in vitro (B and D, original magnification ×200). The concentration of cholesterol ester was measured both in vivo (C, n =5) and in vitro (E). The results represent the mean±SD. * P

Techniques Used: Mouse Assay, Staining, In Vivo, In Vitro, Concentration Assay

CXCL16 siRNA reduced lipid accumulation in HK-2 cells, which consequently decreased ROS production and ECM excretion. HK-2 cells were made quiescent using serum-free medium for 24 h and treated with 30 mmol/L glucose (HG) or 30 mmol/L glucose plus 5 ng/mL IL-1β. Immunofluorescence staining (A, original magnification ×400), real-time PCR (B) and Western blot (C and D) were used to evaluate the expression levels of CXCL16, ADAM10, and CXCR6. Filipin staining was used to examine lipid accumulation in each group (E).
Figure Legend Snippet: CXCL16 siRNA reduced lipid accumulation in HK-2 cells, which consequently decreased ROS production and ECM excretion. HK-2 cells were made quiescent using serum-free medium for 24 h and treated with 30 mmol/L glucose (HG) or 30 mmol/L glucose plus 5 ng/mL IL-1β. Immunofluorescence staining (A, original magnification ×400), real-time PCR (B) and Western blot (C and D) were used to evaluate the expression levels of CXCL16, ADAM10, and CXCR6. Filipin staining was used to examine lipid accumulation in each group (E).

Techniques Used: Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Western Blot, Expressing

3) Product Images from "Lipid Rafts Alter the Stability and Activity of the Cholera Toxin A1 Subunit *"

Article Title: Lipid Rafts Alter the Stability and Activity of the Cholera Toxin A1 Subunit *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.385575

Lipid rafts are required for optimal in vivo activity of cytosolic CTA1. HeLa cells were transfected with a plasmid encoding a CTA1 construct that is expressed directly in the cytosol. Intracellular cAMP levels at 2 and 4 h after transfection were then quantified for untreated and filipin-treated cells. The averages ± S.D. ( error bars ) of 3–4 independent experiments with triplicate samples are shown.
Figure Legend Snippet: Lipid rafts are required for optimal in vivo activity of cytosolic CTA1. HeLa cells were transfected with a plasmid encoding a CTA1 construct that is expressed directly in the cytosol. Intracellular cAMP levels at 2 and 4 h after transfection were then quantified for untreated and filipin-treated cells. The averages ± S.D. ( error bars ) of 3–4 independent experiments with triplicate samples are shown.

Techniques Used: In Vivo, Activity Assay, Transfection, Plasmid Preparation, Construct

Related Articles

Centrifugation:

Article Title: Cell Surface THY-1 Contributes to Human Cytomegalovirus Entry via a Macropinocytosis-Like Process
Article Snippet: Cell culture supernatants from virus-infected cells were centrifuged at 2,000 × g for 30 min at 4°C, and the clarified supernatants were used as virus stocks or further partially purified by centrifugation through a 20% sucrose or sorbitol cushion at 35,000 × g at 4°C for 60 min and resuspended in RPMI 1640 medium with 10% FBS. .. IPA-3 (EMD, Chicago, IL), dynasore monohydrate, and filipin III (Santa Cruz, Santa Cruz, CA), jasplakinolide (Calbiochem, San Diego, CA), 5-( N -ethyl- N -isopropyl)amiloride (EIPA), bafilomycin A1, and monensin (Sigma-Aldrich, St. Louis, MO), and (Cell Signaling Technology, Boston, MA) were used to inhibit virus entry.

Filtration:

Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis
Article Snippet: .. Chemicals and compounds The following compounds were used: 25-hydroxycholesterol (25HC, Sigma-Aldrich, St. Louis, MO), DAPI (Sigma-Aldrich), aminooxybiotin (Biotium, Fremont, CA), aniline hydrochloride (Sigma-Aldrich), avasimibe (Sigma-Aldrich), cholesterol (Sigma-Aldrich), doxycycline (Sigma-Aldrich), filipin III (Santa Cruz Biotechnology, Dallas, TX), gel filtration markers kit (MWGF1000, Sigma-Aldrich), GGTI 298 trifluoroacetate salt hydrate (Sigma-Aldrich), Hygromycin B Gold (InvivoGen, San Diego, CA), IAA (iodoacetamide, Sigma-Aldrich), lipoprotein-depleted fetal bovine serum (LPDS, Kalen Biomedical, Germantown, MD), LMNG (lauryl maltose neopentyl glycol, Anatrace, Maumee, OH), methyl-β-cyclodextrin (MBCD, Sigma-Aldrich), MG132 (Merck Chemicals Ltd, Darmstadt, Germany), N-ethylmaleamide (NEM, Thermo Fisher Scientific), propidium iodide (Sigma-Aldrich), puromycin (Invivogen), SILAC Lys-8-Arg-10 kit (282986444, Silantes GmbH, Munich, Germany), sodium-meta-periodate (Sigma-Aldrich) and ZA (zaragozic acid A, Santa Cruz Biotechnology). .. Antibodies The following antibodies were used in this study: ACC1 (Cell Signaling, Danvers, MA, rabbit pAb, #4190), 1:1000; AlexaFluor R488 anti-mouse IgG (H+L) (Invitrogen, donkey pAb, #A21202), 1:4000 [immunoblotting (IB)], 1:400 [immunofluorescence (IF)]; AlexaFluor R488 anti-rabbit IgG (H+L) (Invitrogen, goat pAb, #A11008), 1:4000 (IB), 1:400 (IF); anti-mouse IgG-HRP (Santa Cruz Biotechnology, goat, #sc-2005), 1:10,000; anti-rabbit IgG-HRP (Santa Cruz Biotechnology, goat, #sc-2030), 1:10,000; EMC1 (rabbit pAb, kind gift of Enilza Espreafico, Sao Paulo, Brazil), 1:1000; EMC2 (Proteintech, Rosemont, IL, rabbit pAb, #25443-1-AP), 1:2000; EMC3 (Abcam, Cambridge, UK, rabbit pAb, #ab175537), 1:500; EMC4 (Abcam, rabbit pAb, #ab123719), 1:1000; EMC5/MMGT1 (Abcam, rabbit pAb, #ab174366), 1:1000; EMC6 (Abcam, rabbit pAb, #ab84902), 1:1000; EMC7 (Abgent, San Diego, CA, rabbit pAb, #AP11145c), 1:500; EMC8 (Abcam, rabbit pAb, #ab180065), 1:500; EMC9 (Abgent, rabbit pAb, #AP5632b), 1:500; EMC10 (Abcam, rabbit pAb, #ab180148), 1:1000; HA (purified from hybridoma, mouse mAb, clone 12CA5), 1:1000; histone H3 (Abcam, rabbit pAb, ab1791), 1:5000; HMGCR (Atlas Antibodies, Bromma, Sweden, mouse mAb, clone CL0260), 1:1000; Hsp70 (Santa Cruz Biotechnology, goat pAb, clone K-20, #sc-1060), 1:1000; LDLR (R & D Systems, Minneapolis, MN, goat pAb, #AF2148), 1:1000; Rap1a (Santa Cruz Biotechnology, rabbit pAb, clone C-17, #sc-1482), 1:1000; SCD1 (Abcam, mouse mAb, clone CD.E10, #ab19862), 1:1000; SM (Proteintech, rabbit pAb, #12544-1-AP), 1:1000; SOAT1 (Santa Cruz Biotechnology, mouse mAb, clone A-11, #sc-136959), 1:1000; SQS (Abcam, rabbit mAb, #ab109723), 1:2000; SQS (Abcam, rabbit mAb, #ab195046), 1:200 (IF), 1:2000 (WB); SREBP2 (R & D Systems, goat pAb, #AF7119), 1:1000; tubulin (Sigma-Aldrich, mouse mAb, #T5168/b-5-1-2), 1:1000; and ubiquitin (Cell Signaling Technologies, rabbit pAb, #3933S), 1:1000.

In Vitro:

Article Title: Caveolin-1 mediates cellular distribution of HER2 and affects trastuzumab binding and therapeutic efficacy
Article Snippet: To inhibit caveolae-mediated pathways, cells were incubated with 2.5 mM MβCD or 0.5 μg mL−1 filipin (Santa Cruz Biotechnology) for 30 min before addition of trastuzumab. .. For in vitro experiments with lovastatin, cells were incubated with 25 μM of the active form of lovastatin (Millipore) for 4 h prior addition of trastuzumab .

Article Title: Lipid Rafts Alter the Stability and Activity of the Cholera Toxin A1 Subunit *
Article Snippet: Filipin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA); CT was purchased from List Biologicals (Campbell, CA); and isoproterenol was purchased from Sigma-Aldrich. .. CTA1 with a C-terminal His6 tag was purified as described previously ( ) and used for all in vitro studies.

Radio Immunoprecipitation:

Article Title: Autophagy regulates testosterone synthesis by facilitating cholesterol uptake in Leydig cells
Article Snippet: Percoll (sc-296039) and Filipin III (sc-205323) were purchased from Santa Cruz Biotechnology, Inc. HDL labeled with DiI-HDL (H8910) was purchased from Solarbio. .. Radioimmunoprecipitation assay (RIPA) buffer (P0013C) and bicinchoninic acid kits (P0012) were purchased from Beyotime.

Indirect Immunoperoxidase Assay:

Article Title: Cell Surface THY-1 Contributes to Human Cytomegalovirus Entry via a Macropinocytosis-Like Process
Article Snippet: .. IPA-3 (EMD, Chicago, IL), dynasore monohydrate, and filipin III (Santa Cruz, Santa Cruz, CA), jasplakinolide (Calbiochem, San Diego, CA), 5-( N -ethyl- N -isopropyl)amiloride (EIPA), bafilomycin A1, and monensin (Sigma-Aldrich, St. Louis, MO), and (Cell Signaling Technology, Boston, MA) were used to inhibit virus entry. .. Cells were grown and infected on silicon chips, fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, and then postfixed with 1.0% osmium tetroxide in 0.1 M sodium cacodylate buffer.

Mouse Assay:

Article Title: Inflammation-activated CXCL16 pathway contributes to tubulointerstitial injury in mouse diabetic nephropathy
Article Snippet: Lipid accumulation in HK-2 cells or in the kidneys of db/db mice was evaluated by Filipin staining. .. Briefly, samples were fixed with 4% paraformaldehyde and then stained with Filipin (50 ng/mL, Santa Cruz, Dallas, USA) for 30 min at room temperature.

Flow Cytometry:

Article Title: Multifunctional quantum dot DNA hydrogels
Article Snippet: Paragraph title: Cellular uptake by flow cytometry ... For the endocytic uptake pathway determination, cells were pretreated with the following endocytosis inhibitors 1 h prior to QDH treatment: 200 μM dynasore, 4 μM filipin III, or 400 μM cytochalasin D (Santa Cruz Biotechnology).

Microscopy:

Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis
Article Snippet: Cells were incubated with Filipin III (Santa Cruz Biotechnology, 25 µg/ml in PBS, 30 min) and rinsed three times with PBS. .. Antibody-bound proteins were visualised at an excitation wavelength of 488 nm and DAPI and Filipin III were imaged at 405 nm using an LSM 710 (Zeiss, Oberkochen, Germany) confocal microscope.

Article Title: Inflammation-activated CXCL16 pathway contributes to tubulointerstitial injury in mouse diabetic nephropathy
Article Snippet: Briefly, samples were fixed with 4% paraformaldehyde and then stained with Filipin (50 ng/mL, Santa Cruz, Dallas, USA) for 30 min at room temperature. .. The results of Filipin staining were examined by laser microscopy (×200).

Purification:

Article Title: Cell Surface THY-1 Contributes to Human Cytomegalovirus Entry via a Macropinocytosis-Like Process
Article Snippet: Cell culture supernatants from virus-infected cells were centrifuged at 2,000 × g for 30 min at 4°C, and the clarified supernatants were used as virus stocks or further partially purified by centrifugation through a 20% sucrose or sorbitol cushion at 35,000 × g at 4°C for 60 min and resuspended in RPMI 1640 medium with 10% FBS. .. IPA-3 (EMD, Chicago, IL), dynasore monohydrate, and filipin III (Santa Cruz, Santa Cruz, CA), jasplakinolide (Calbiochem, San Diego, CA), 5-( N -ethyl- N -isopropyl)amiloride (EIPA), bafilomycin A1, and monensin (Sigma-Aldrich, St. Louis, MO), and (Cell Signaling Technology, Boston, MA) were used to inhibit virus entry.

Article Title: Utilization of HEPES for Enhancing Protein Transfection into Mammalian Cells
Article Snippet: The procedures used for the production and purification of the v5-tagged rhSTIP1 proteins have been previously described in detail. .. Sucrose and HEPES were from Sigma, filipin was obtained from Santa Cruz Biotechnology, and amiloride hydrochloride was purchased from Toronto Research Chemicals (Toronto, ON, Canada).

Article Title: Lipid Rafts Alter the Stability and Activity of the Cholera Toxin A1 Subunit *
Article Snippet: Filipin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA); CT was purchased from List Biologicals (Campbell, CA); and isoproterenol was purchased from Sigma-Aldrich. .. CTA1 with a C-terminal His6 tag was purified as described previously ( ) and used for all in vitro studies.

Cytometry:

Article Title: Multifunctional quantum dot DNA hydrogels
Article Snippet: Paragraph title: Cellular uptake by flow cytometry ... For the endocytic uptake pathway determination, cells were pretreated with the following endocytosis inhibitors 1 h prior to QDH treatment: 200 μM dynasore, 4 μM filipin III, or 400 μM cytochalasin D (Santa Cruz Biotechnology).

FLAG-tag:

Article Title: Utilization of HEPES for Enhancing Protein Transfection into Mammalian Cells
Article Snippet: The FLAG-Rev peptide consists of a FLAG tag and the Rev peptide, which is a 13-amino-acid fragment of the HIV and has high affinity with the B23 protein. .. Sucrose and HEPES were from Sigma, filipin was obtained from Santa Cruz Biotechnology, and amiloride hydrochloride was purchased from Toronto Research Chemicals (Toronto, ON, Canada).

Incubation:

Article Title: Caveolin-1 mediates cellular distribution of HER2 and affects trastuzumab binding and therapeutic efficacy
Article Snippet: .. To inhibit caveolae-mediated pathways, cells were incubated with 2.5 mM MβCD or 0.5 μg mL−1 filipin (Santa Cruz Biotechnology) for 30 min before addition of trastuzumab. .. For in vitro experiments with lovastatin, cells were incubated with 25 μM of the active form of lovastatin (Millipore) for 4 h prior addition of trastuzumab .

Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis
Article Snippet: .. Cells were incubated with Filipin III (Santa Cruz Biotechnology, 25 µg/ml in PBS, 30 min) and rinsed three times with PBS. ..

Cell Culture:

Article Title: Cell Surface THY-1 Contributes to Human Cytomegalovirus Entry via a Macropinocytosis-Like Process
Article Snippet: Cell culture supernatants from virus-infected cells were centrifuged at 2,000 × g for 30 min at 4°C, and the clarified supernatants were used as virus stocks or further partially purified by centrifugation through a 20% sucrose or sorbitol cushion at 35,000 × g at 4°C for 60 min and resuspended in RPMI 1640 medium with 10% FBS. .. IPA-3 (EMD, Chicago, IL), dynasore monohydrate, and filipin III (Santa Cruz, Santa Cruz, CA), jasplakinolide (Calbiochem, San Diego, CA), 5-( N -ethyl- N -isopropyl)amiloride (EIPA), bafilomycin A1, and monensin (Sigma-Aldrich, St. Louis, MO), and (Cell Signaling Technology, Boston, MA) were used to inhibit virus entry.

Labeling:

Article Title: Autophagy regulates testosterone synthesis by facilitating cholesterol uptake in Leydig cells
Article Snippet: .. Percoll (sc-296039) and Filipin III (sc-205323) were purchased from Santa Cruz Biotechnology, Inc. HDL labeled with DiI-HDL (H8910) was purchased from Solarbio. .. Radioimmunoprecipitation assay (RIPA) buffer (P0013C) and bicinchoninic acid kits (P0012) were purchased from Beyotime.

Confocal Microscopy:

Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis
Article Snippet: Paragraph title: Indirect immunofluorescence confocal microscopy ... Cells were incubated with Filipin III (Santa Cruz Biotechnology, 25 µg/ml in PBS, 30 min) and rinsed three times with PBS.

Staining:

Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis
Article Snippet: For Filipin III staining, after fixation with 4% PFA, cells were rinsed three times with PBS before quenching residual PFA with 1.5 mg/ml glycine plus PBS (10 min). .. Cells were incubated with Filipin III (Santa Cruz Biotechnology, 25 µg/ml in PBS, 30 min) and rinsed three times with PBS.

Article Title: Inflammation-activated CXCL16 pathway contributes to tubulointerstitial injury in mouse diabetic nephropathy
Article Snippet: .. Briefly, samples were fixed with 4% paraformaldehyde and then stained with Filipin (50 ng/mL, Santa Cruz, Dallas, USA) for 30 min at room temperature. .. The results of Filipin staining were examined by laser microscopy (×200).

Recombinant:

Article Title: Utilization of HEPES for Enhancing Protein Transfection into Mammalian Cells
Article Snippet: Paragraph title: Antibodies, Recombinant Proteins, Peptides, and Chemicals ... Sucrose and HEPES were from Sigma, filipin was obtained from Santa Cruz Biotechnology, and amiloride hydrochloride was purchased from Toronto Research Chemicals (Toronto, ON, Canada).

Fluorescence In Situ Hybridization:

Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis
Article Snippet: Indirect immunofluorescence confocal microscopy Cells seeded on 12-mm glass coverslips (Nunc) were washed twice with PBS and fixed with 4% paraformaldehyde (PFA) for 20 min. PFA was removed by rinsing twice with PBS and cells were permeabilised with 0.1% (v/v) Triton X-100 in PBS (5 min), washed twice with PBS, and incubated in blocking buffer [0.2% (w/v) fish skin gelatin (FSG) plus PBS, 60 min]. .. Cells were incubated with Filipin III (Santa Cruz Biotechnology, 25 µg/ml in PBS, 30 min) and rinsed three times with PBS.

Immunofluorescence:

Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis
Article Snippet: Paragraph title: Indirect immunofluorescence confocal microscopy ... Cells were incubated with Filipin III (Santa Cruz Biotechnology, 25 µg/ml in PBS, 30 min) and rinsed three times with PBS.

Blocking Assay:

Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis
Article Snippet: Primary antibody [anti-SQS rabbit mAb, ab195046 (Abcam), 1:200] in blocking buffer was added (60 min), the coverslips were rinsed twice with PBS and incubated in the dark (60 min) with Alexa Fluor 488-conjugated secondary antibody (1:400 in blocking buffer). .. Cells were incubated with Filipin III (Santa Cruz Biotechnology, 25 µg/ml in PBS, 30 min) and rinsed three times with PBS.

Software:

Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis
Article Snippet: Cells were incubated with Filipin III (Santa Cruz Biotechnology, 25 µg/ml in PBS, 30 min) and rinsed three times with PBS. .. Images were processed using ImageJ and Adobe Illustrator software.

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    Santa Cruz Biotechnology filipin iii
    EMC loss does not activate a cholesterol homeostatic response. (A) Isolation of cytoplasmic (C) and nuclear (N) fractions from WT and ΔEMC6 (Δ6) cells by subcellular fractionation. Cells were treated with MBCD (1 mM), 25-hydroxycholesterol (25HC, 25 µM) or ethanol (control, Veh.) for 4 h. Western blots probing for the full-length (FL) and cleaved N-terminal fragment (N) of SREBP-2 are shown, along with Sec61β and histone H3 as controls for cytoplasmic and nuclear fractions, respectively. The asterisk (*) denotes a nonspecific band. (B) mRNA levels of HMGCR, SQS and SM as determined by quantitative real-time RT-PCR (qRT-PCR). Transcript levels present in untreated ΔEMC5 (Δ5) and Δ6 U2OS Flp-In™ T-Rex™ cells relative to WT are shown as means±s.d. ( n =6). (C) Western blots of whole-cell lysates derived from WT, Δ5 and Δ6 cells grown in normal medium were probed with the indicated antibodies, where Hsp70 served as a loading control. (D) <t>Filipin</t> <t>III</t> staining of WT and Δ6 cells maintained in DMEM plus 10% FCS to monitor intracellular cholesterol distribution. (E) mRNA levels of HMGCR, SM and SQS from WT and Δ6 cells treated with 1 mM MBCD for 0, 2 and 4 h, as determined by qRT-PCR relative to WT. Means±s.d. are shown ( n =3).
    Filipin Iii, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/filipin iii/product/Santa Cruz Biotechnology
    Average 95 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    filipin iii - by Bioz Stars, 2020-04
    95/100 stars
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    EMC loss does not activate a cholesterol homeostatic response. (A) Isolation of cytoplasmic (C) and nuclear (N) fractions from WT and ΔEMC6 (Δ6) cells by subcellular fractionation. Cells were treated with MBCD (1 mM), 25-hydroxycholesterol (25HC, 25 µM) or ethanol (control, Veh.) for 4 h. Western blots probing for the full-length (FL) and cleaved N-terminal fragment (N) of SREBP-2 are shown, along with Sec61β and histone H3 as controls for cytoplasmic and nuclear fractions, respectively. The asterisk (*) denotes a nonspecific band. (B) mRNA levels of HMGCR, SQS and SM as determined by quantitative real-time RT-PCR (qRT-PCR). Transcript levels present in untreated ΔEMC5 (Δ5) and Δ6 U2OS Flp-In™ T-Rex™ cells relative to WT are shown as means±s.d. ( n =6). (C) Western blots of whole-cell lysates derived from WT, Δ5 and Δ6 cells grown in normal medium were probed with the indicated antibodies, where Hsp70 served as a loading control. (D) Filipin III staining of WT and Δ6 cells maintained in DMEM plus 10% FCS to monitor intracellular cholesterol distribution. (E) mRNA levels of HMGCR, SM and SQS from WT and Δ6 cells treated with 1 mM MBCD for 0, 2 and 4 h, as determined by qRT-PCR relative to WT. Means±s.d. are shown ( n =3).

    Journal: Journal of Cell Science

    Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis

    doi: 10.1242/jcs.223453

    Figure Lengend Snippet: EMC loss does not activate a cholesterol homeostatic response. (A) Isolation of cytoplasmic (C) and nuclear (N) fractions from WT and ΔEMC6 (Δ6) cells by subcellular fractionation. Cells were treated with MBCD (1 mM), 25-hydroxycholesterol (25HC, 25 µM) or ethanol (control, Veh.) for 4 h. Western blots probing for the full-length (FL) and cleaved N-terminal fragment (N) of SREBP-2 are shown, along with Sec61β and histone H3 as controls for cytoplasmic and nuclear fractions, respectively. The asterisk (*) denotes a nonspecific band. (B) mRNA levels of HMGCR, SQS and SM as determined by quantitative real-time RT-PCR (qRT-PCR). Transcript levels present in untreated ΔEMC5 (Δ5) and Δ6 U2OS Flp-In™ T-Rex™ cells relative to WT are shown as means±s.d. ( n =6). (C) Western blots of whole-cell lysates derived from WT, Δ5 and Δ6 cells grown in normal medium were probed with the indicated antibodies, where Hsp70 served as a loading control. (D) Filipin III staining of WT and Δ6 cells maintained in DMEM plus 10% FCS to monitor intracellular cholesterol distribution. (E) mRNA levels of HMGCR, SM and SQS from WT and Δ6 cells treated with 1 mM MBCD for 0, 2 and 4 h, as determined by qRT-PCR relative to WT. Means±s.d. are shown ( n =3).

    Article Snippet: Chemicals and compounds The following compounds were used: 25-hydroxycholesterol (25HC, Sigma-Aldrich, St. Louis, MO), DAPI (Sigma-Aldrich), aminooxybiotin (Biotium, Fremont, CA), aniline hydrochloride (Sigma-Aldrich), avasimibe (Sigma-Aldrich), cholesterol (Sigma-Aldrich), doxycycline (Sigma-Aldrich), filipin III (Santa Cruz Biotechnology, Dallas, TX), gel filtration markers kit (MWGF1000, Sigma-Aldrich), GGTI 298 trifluoroacetate salt hydrate (Sigma-Aldrich), Hygromycin B Gold (InvivoGen, San Diego, CA), IAA (iodoacetamide, Sigma-Aldrich), lipoprotein-depleted fetal bovine serum (LPDS, Kalen Biomedical, Germantown, MD), LMNG (lauryl maltose neopentyl glycol, Anatrace, Maumee, OH), methyl-β-cyclodextrin (MBCD, Sigma-Aldrich), MG132 (Merck Chemicals Ltd, Darmstadt, Germany), N-ethylmaleamide (NEM, Thermo Fisher Scientific), propidium iodide (Sigma-Aldrich), puromycin (Invivogen), SILAC Lys-8-Arg-10 kit (282986444, Silantes GmbH, Munich, Germany), sodium-meta-periodate (Sigma-Aldrich) and ZA (zaragozic acid A, Santa Cruz Biotechnology).

    Techniques: Isolation, Fractionation, Western Blot, Quantitative RT-PCR, Derivative Assay, Staining

    HEPES-Induced Protein Transfection Was Accomplished through Endocytosis and Charge Neutralization (A–C) MDAH2774 cells were pretreated for 30 min with Opti-MEM (Ctrl), 0.4 M sucrose (an inhibitor of clathrin-mediated endocytosis), 5 μg/mL filipin (an inhibitor of caveolae-mediated endocytosis), or 0.25 mM amiloride (an inhibitor of macropinocytosis). Alexa Fluor 488-conjugated antibodies were delivered into cells using 20 mM HEPES, as described in The HEPES Method , followed by a 4-h incubation period. (A) Transfection efficiency was determined using a confocal fluorescent microscope. (B) Alexa Fluor 488 fluorescent intensity signals were quantified using Q-Win software and are expressed as relative ratios. (C) At 4 h of inhibitor treatment, cytotoxicity was analyzed using the LDH assay. Error bars indicate the SEM (n = 3).*p

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Utilization of HEPES for Enhancing Protein Transfection into Mammalian Cells

    doi: 10.1016/j.omtm.2018.12.005

    Figure Lengend Snippet: HEPES-Induced Protein Transfection Was Accomplished through Endocytosis and Charge Neutralization (A–C) MDAH2774 cells were pretreated for 30 min with Opti-MEM (Ctrl), 0.4 M sucrose (an inhibitor of clathrin-mediated endocytosis), 5 μg/mL filipin (an inhibitor of caveolae-mediated endocytosis), or 0.25 mM amiloride (an inhibitor of macropinocytosis). Alexa Fluor 488-conjugated antibodies were delivered into cells using 20 mM HEPES, as described in The HEPES Method , followed by a 4-h incubation period. (A) Transfection efficiency was determined using a confocal fluorescent microscope. (B) Alexa Fluor 488 fluorescent intensity signals were quantified using Q-Win software and are expressed as relative ratios. (C) At 4 h of inhibitor treatment, cytotoxicity was analyzed using the LDH assay. Error bars indicate the SEM (n = 3).*p

    Article Snippet: Sucrose and HEPES were from Sigma, filipin was obtained from Santa Cruz Biotechnology, and amiloride hydrochloride was purchased from Toronto Research Chemicals (Toronto, ON, Canada).

    Techniques: Transfection, Neutralization, Incubation, Microscopy, Software, Lactate Dehydrogenase Assay

    Inflammation increased lipid accumulation to accelerate tubulointerstitial injuries. Eight-week-old diabetic db/db mice were randomly assigned and received subcutaneous injections with either 0.5 mL distilled water ( db/db , n =10) or 0.5 mL 10% casein ( db/db +casein, n =10) every other day for 8 weeks. HK-2 cells were made quiescent using serum-free medium for 24 h, and treated with 30 mmol/L glucose (HG) or 30 mmol/L glucose plus 5 ng/mL IL-1β. TG, triglyceride; TC, total cholesterol; LDL, low-density lipoprotein; HDL, high-density lipoprotein (A). Filipin staining was used to examine lipid accumulation both in vivo and in vitro (B and D, original magnification ×200). The concentration of cholesterol ester was measured both in vivo (C, n =5) and in vitro (E). The results represent the mean±SD. * P

    Journal: Acta Pharmacologica Sinica

    Article Title: Inflammation-activated CXCL16 pathway contributes to tubulointerstitial injury in mouse diabetic nephropathy

    doi: 10.1038/aps.2017.177

    Figure Lengend Snippet: Inflammation increased lipid accumulation to accelerate tubulointerstitial injuries. Eight-week-old diabetic db/db mice were randomly assigned and received subcutaneous injections with either 0.5 mL distilled water ( db/db , n =10) or 0.5 mL 10% casein ( db/db +casein, n =10) every other day for 8 weeks. HK-2 cells were made quiescent using serum-free medium for 24 h, and treated with 30 mmol/L glucose (HG) or 30 mmol/L glucose plus 5 ng/mL IL-1β. TG, triglyceride; TC, total cholesterol; LDL, low-density lipoprotein; HDL, high-density lipoprotein (A). Filipin staining was used to examine lipid accumulation both in vivo and in vitro (B and D, original magnification ×200). The concentration of cholesterol ester was measured both in vivo (C, n =5) and in vitro (E). The results represent the mean±SD. * P

    Article Snippet: Briefly, samples were fixed with 4% paraformaldehyde and then stained with Filipin (50 ng/mL, Santa Cruz, Dallas, USA) for 30 min at room temperature.

    Techniques: Mouse Assay, Staining, In Vivo, In Vitro, Concentration Assay

    CXCL16 siRNA reduced lipid accumulation in HK-2 cells, which consequently decreased ROS production and ECM excretion. HK-2 cells were made quiescent using serum-free medium for 24 h and treated with 30 mmol/L glucose (HG) or 30 mmol/L glucose plus 5 ng/mL IL-1β. Immunofluorescence staining (A, original magnification ×400), real-time PCR (B) and Western blot (C and D) were used to evaluate the expression levels of CXCL16, ADAM10, and CXCR6. Filipin staining was used to examine lipid accumulation in each group (E).

    Journal: Acta Pharmacologica Sinica

    Article Title: Inflammation-activated CXCL16 pathway contributes to tubulointerstitial injury in mouse diabetic nephropathy

    doi: 10.1038/aps.2017.177

    Figure Lengend Snippet: CXCL16 siRNA reduced lipid accumulation in HK-2 cells, which consequently decreased ROS production and ECM excretion. HK-2 cells were made quiescent using serum-free medium for 24 h and treated with 30 mmol/L glucose (HG) or 30 mmol/L glucose plus 5 ng/mL IL-1β. Immunofluorescence staining (A, original magnification ×400), real-time PCR (B) and Western blot (C and D) were used to evaluate the expression levels of CXCL16, ADAM10, and CXCR6. Filipin staining was used to examine lipid accumulation in each group (E).

    Article Snippet: Briefly, samples were fixed with 4% paraformaldehyde and then stained with Filipin (50 ng/mL, Santa Cruz, Dallas, USA) for 30 min at room temperature.

    Techniques: Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Western Blot, Expressing

    Lipid rafts are required for optimal in vivo activity of cytosolic CTA1. HeLa cells were transfected with a plasmid encoding a CTA1 construct that is expressed directly in the cytosol. Intracellular cAMP levels at 2 and 4 h after transfection were then quantified for untreated and filipin-treated cells. The averages ± S.D. ( error bars ) of 3–4 independent experiments with triplicate samples are shown.

    Journal: The Journal of Biological Chemistry

    Article Title: Lipid Rafts Alter the Stability and Activity of the Cholera Toxin A1 Subunit *

    doi: 10.1074/jbc.M112.385575

    Figure Lengend Snippet: Lipid rafts are required for optimal in vivo activity of cytosolic CTA1. HeLa cells were transfected with a plasmid encoding a CTA1 construct that is expressed directly in the cytosol. Intracellular cAMP levels at 2 and 4 h after transfection were then quantified for untreated and filipin-treated cells. The averages ± S.D. ( error bars ) of 3–4 independent experiments with triplicate samples are shown.

    Article Snippet: Filipin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA); CT was purchased from List Biologicals (Campbell, CA); and isoproterenol was purchased from Sigma-Aldrich.

    Techniques: In Vivo, Activity Assay, Transfection, Plasmid Preparation, Construct