lysis buffer  (Roche)


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    Structured Review

    Roche lysis buffer
    Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lysis buffer/product/Roche
    Average 88 stars, based on 40 article reviews
    Price from $9.99 to $1999.99
    lysis buffer - by Bioz Stars, 2022-10
    88/100 stars

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    Roche immunoprecipitation lysis buffer
    MAGE-A11 stabilizes E2F1 and mediates an interaction between E2F1 and p107. A , pCMV5 (1 μg) ( lane 1 ), 1 μg of pSG5-MAGE ( lanes 2 and 3 ), 0.5 μg of CMV-E2F1 ( lanes 4 and 5 ), or 1 μg of pSG5-MAGE and 0.5 μg of CMV-E2F1 together ( lanes 6 and 7 ) was expressed in COS1 cells. Cells were incubated for 24 h before harvest in serum-free medium with or without 0.1 μg/ml EGF. Cell extracts (40 μg of protein/lane) were probed on the transblot using E2F1 (sc-193) and MAGE-A11 antibodies. B , pCMV-FLAG (4 μg) (−) or 4 μg of pCMV-FLAG-p107 was expressed in COS1 cells with or without 3 μg of pSG5-MAGE or 4 μg of CMV-E2F1 alone or together. Cells were incubated for 24 h in serum-free medium with 0.1 μg/ml EGF and immunoprecipitated using FLAG affinity resin. Transblots of cell extracts (40 μg of protein/lane, left ) or immunoprecipitates ( IP , right ) were probed using p107, E2F1 (sc-193), and MAGE-A11 antibodies. C , pCMV-FLAG, or pCMV-FLAG-p107 (4 μg/dish) was expressed in three 10-cm dishes plated at 7 × 10 6 LAPC-4 cells/dish using 8 μl of X-tremeGENE in 160 μl of medium added/dish. The next day, cells were transferred to serum-free medium with 0.1 μg/ml EGF and incubated for 24 h. The cell extract (40 μg of protein/lane) prepared in <t>immunoprecipitation</t> lysis buffer and immunoprecipitates were probed overnight at 4 °C on transblots using p107 (sc-318, 1:200 dilution) and MAGE-A11 antibodies (15 μg/ml). The blot was stripped and reprobed using E2F1 (sc-193) antibody (1:100 dilution).
    Immunoprecipitation Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/immunoprecipitation lysis buffer/product/Roche
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    immunoprecipitation lysis buffer - by Bioz Stars, 2022-10
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    90
    Roche sds lysis buffer
    Functional characterization of modulation of LRRK2 at T1410. (A) A LanthaScreen assay was used to assess LRRK2 T1410A kinase activity. Titration of wildtype, G2019S, and T1410A (G2019S) LRRK2 proteins demonstrate that mutation T1410A does not affect phosphorylation of LRRKtide. (B) To investigate dimer formation HEK-293T cells were transfected with the indicated LRRK2 constructs. Cell pellets were split equally for lysis by freeze/thaw cycles directly in PBS or lysis with 1% <t>SDS</t> and PBS with sonication, and 10 ug of protein lysate was loaded onto a native gel (3–12% <t>Bis-Tris)</t> or an SDS gel (7% Tris acetate-SDS), respectively. LRRK2 complexes were visualized with the anti-HA antibody by Western blot with standard ECL. Dimer-sized structures are evident (signal from 480 to 550 kDa), monomeric LRRK2 (278 kDa) is only visible by overexposure (Supplemental Figure S1 , [25] ).Western blots representative of five independent experiments are shown. Normalization of the LRRK2 dimer to total LRRK2 protein (SDS-solubilized) was done using densitometry analysis. ** p
    Sds Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sds lysis buffer/product/Roche
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    MAGE-A11 stabilizes E2F1 and mediates an interaction between E2F1 and p107. A , pCMV5 (1 μg) ( lane 1 ), 1 μg of pSG5-MAGE ( lanes 2 and 3 ), 0.5 μg of CMV-E2F1 ( lanes 4 and 5 ), or 1 μg of pSG5-MAGE and 0.5 μg of CMV-E2F1 together ( lanes 6 and 7 ) was expressed in COS1 cells. Cells were incubated for 24 h before harvest in serum-free medium with or without 0.1 μg/ml EGF. Cell extracts (40 μg of protein/lane) were probed on the transblot using E2F1 (sc-193) and MAGE-A11 antibodies. B , pCMV-FLAG (4 μg) (−) or 4 μg of pCMV-FLAG-p107 was expressed in COS1 cells with or without 3 μg of pSG5-MAGE or 4 μg of CMV-E2F1 alone or together. Cells were incubated for 24 h in serum-free medium with 0.1 μg/ml EGF and immunoprecipitated using FLAG affinity resin. Transblots of cell extracts (40 μg of protein/lane, left ) or immunoprecipitates ( IP , right ) were probed using p107, E2F1 (sc-193), and MAGE-A11 antibodies. C , pCMV-FLAG, or pCMV-FLAG-p107 (4 μg/dish) was expressed in three 10-cm dishes plated at 7 × 10 6 LAPC-4 cells/dish using 8 μl of X-tremeGENE in 160 μl of medium added/dish. The next day, cells were transferred to serum-free medium with 0.1 μg/ml EGF and incubated for 24 h. The cell extract (40 μg of protein/lane) prepared in immunoprecipitation lysis buffer and immunoprecipitates were probed overnight at 4 °C on transblots using p107 (sc-318, 1:200 dilution) and MAGE-A11 antibodies (15 μg/ml). The blot was stripped and reprobed using E2F1 (sc-193) antibody (1:100 dilution).

    Journal: The Journal of Biological Chemistry

    Article Title: Proto-oncogene Activity of Melanoma Antigen-A11 (MAGE-A11) Regulates Retinoblastoma-related p107 and E2F1 Proteins *

    doi: 10.1074/jbc.M113.468579

    Figure Lengend Snippet: MAGE-A11 stabilizes E2F1 and mediates an interaction between E2F1 and p107. A , pCMV5 (1 μg) ( lane 1 ), 1 μg of pSG5-MAGE ( lanes 2 and 3 ), 0.5 μg of CMV-E2F1 ( lanes 4 and 5 ), or 1 μg of pSG5-MAGE and 0.5 μg of CMV-E2F1 together ( lanes 6 and 7 ) was expressed in COS1 cells. Cells were incubated for 24 h before harvest in serum-free medium with or without 0.1 μg/ml EGF. Cell extracts (40 μg of protein/lane) were probed on the transblot using E2F1 (sc-193) and MAGE-A11 antibodies. B , pCMV-FLAG (4 μg) (−) or 4 μg of pCMV-FLAG-p107 was expressed in COS1 cells with or without 3 μg of pSG5-MAGE or 4 μg of CMV-E2F1 alone or together. Cells were incubated for 24 h in serum-free medium with 0.1 μg/ml EGF and immunoprecipitated using FLAG affinity resin. Transblots of cell extracts (40 μg of protein/lane, left ) or immunoprecipitates ( IP , right ) were probed using p107, E2F1 (sc-193), and MAGE-A11 antibodies. C , pCMV-FLAG, or pCMV-FLAG-p107 (4 μg/dish) was expressed in three 10-cm dishes plated at 7 × 10 6 LAPC-4 cells/dish using 8 μl of X-tremeGENE in 160 μl of medium added/dish. The next day, cells were transferred to serum-free medium with 0.1 μg/ml EGF and incubated for 24 h. The cell extract (40 μg of protein/lane) prepared in immunoprecipitation lysis buffer and immunoprecipitates were probed overnight at 4 °C on transblots using p107 (sc-318, 1:200 dilution) and MAGE-A11 antibodies (15 μg/ml). The blot was stripped and reprobed using E2F1 (sc-193) antibody (1:100 dilution).

    Article Snippet: Cell pellets were extracted in immunoprecipitation lysis buffer containing 1% Triton X-100, 0.15 m NaCl, 50 m m NaF, 2 m m sodium vanadate, 2 m m EDTA, 50 m m Tris (pH 7.6), 1 m m phenylmethylsulfonyl fluoride, 1 m m dithiothreitol, and complete protease inhibitors (Roche Applied Science) with or without 0.5% deoxycholate or 10% glycerol.

    Techniques: Incubation, Immunoprecipitation, Lysis

    MAGE-A11 interacts with p107 and Rb but not p130. pCMV-FLAG (−), pCMV-FLAG-MAGE, or FLAG-MAGE-(112–429) (5 μg) was expressed in COS1 cells with 5 μg of CMV-p107 ( A ), pCMV-hRb ( B ), or pcDNA3-p130 ( C ). Cells were incubated for 24 h in serum-free medium containing 0.1 μg/ml EGF and harvested in immunoprecipitation lysis buffer without deoxycholate or glycerol. Cell extracts (50 μg of protein/lane, right ) and immunoprecipitates ( IP , left ) were probed using p107, Rb, p130, and MAGE-A11 antibodies. Arrows , p107, Rb, p130, FLAG-MAGE, and IgG.

    Journal: The Journal of Biological Chemistry

    Article Title: Proto-oncogene Activity of Melanoma Antigen-A11 (MAGE-A11) Regulates Retinoblastoma-related p107 and E2F1 Proteins *

    doi: 10.1074/jbc.M113.468579

    Figure Lengend Snippet: MAGE-A11 interacts with p107 and Rb but not p130. pCMV-FLAG (−), pCMV-FLAG-MAGE, or FLAG-MAGE-(112–429) (5 μg) was expressed in COS1 cells with 5 μg of CMV-p107 ( A ), pCMV-hRb ( B ), or pcDNA3-p130 ( C ). Cells were incubated for 24 h in serum-free medium containing 0.1 μg/ml EGF and harvested in immunoprecipitation lysis buffer without deoxycholate or glycerol. Cell extracts (50 μg of protein/lane, right ) and immunoprecipitates ( IP , left ) were probed using p107, Rb, p130, and MAGE-A11 antibodies. Arrows , p107, Rb, p130, FLAG-MAGE, and IgG.

    Article Snippet: Cell pellets were extracted in immunoprecipitation lysis buffer containing 1% Triton X-100, 0.15 m NaCl, 50 m m NaF, 2 m m sodium vanadate, 2 m m EDTA, 50 m m Tris (pH 7.6), 1 m m phenylmethylsulfonyl fluoride, 1 m m dithiothreitol, and complete protease inhibitors (Roche Applied Science) with or without 0.5% deoxycholate or 10% glycerol.

    Techniques: Incubation, Immunoprecipitation, Lysis

    ERBIN expression is reduced in STAT3 mut patient cells and in newly identified individuals with an ERBB2IP mutation. (A, top) Representative immunoblot showing ERBIN expression in primary dermal fibroblasts from control, STAT3 mut , and ERBB2IP mut patients. Lines indicate rearranged segments of one blot. (Bottom) Integrated densitometry of combined data comparing control ( n = 4) with STAT3 mut (SH2 mutants, n = 2; DNA-binding mutants, n = 2) or ERBB2IP mut ( n = 2) individuals. (B) ERBIN expression in naive (CD45RO − ) or memory (CD45RO + ) CD4 T cells from control ( n = 15), STAT3 mut ( n = 17), and ERBB2IP mut ( n = 3) patients. (C) Pedigree, chromatogram, and schematic of mutation (c.1588G > T p.[D530Y]) in ERBB2IP identified in a family with a dominant congenital allergic disorder with connective tissue features. LRR, leucine rich repeat domain; PDZ, PSD95-Dlg1-zo-1–like domain; SSID, SMAD–SARA interacting domain. (D) Representative immunoblot of GFP after MYC immunoprecipitation (IP) in 293T cells in which MYC-tagged wild-type STAT3 (MYC- STAT3 WT ) and GFP-tagged ERBIN-mutant (GFP- ERBB2IP mut ) or wild-type (GFP- ERBB2IP WT ) constructs were overexpressed in the presence or absence of STAT3 activation by IL-6. (E) Effect of wild-type ERBIN ( ERBB2IP WT ) or mutant ERBIN ( ERBB2IP mut ) on TGF-β–induced SMAD-reporter activity. AU, arbitrary units. Data are representative or combined from at least three independent experiments and represented as the mean ± SEM. Paired and unpaired two-tailed Student’s t tests and Mann-Whitney tests were used where appropriate. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: ERBIN deficiency links STAT3 and TGF-β pathway defects with atopy in humans

    doi: 10.1084/jem.20161435

    Figure Lengend Snippet: ERBIN expression is reduced in STAT3 mut patient cells and in newly identified individuals with an ERBB2IP mutation. (A, top) Representative immunoblot showing ERBIN expression in primary dermal fibroblasts from control, STAT3 mut , and ERBB2IP mut patients. Lines indicate rearranged segments of one blot. (Bottom) Integrated densitometry of combined data comparing control ( n = 4) with STAT3 mut (SH2 mutants, n = 2; DNA-binding mutants, n = 2) or ERBB2IP mut ( n = 2) individuals. (B) ERBIN expression in naive (CD45RO − ) or memory (CD45RO + ) CD4 T cells from control ( n = 15), STAT3 mut ( n = 17), and ERBB2IP mut ( n = 3) patients. (C) Pedigree, chromatogram, and schematic of mutation (c.1588G > T p.[D530Y]) in ERBB2IP identified in a family with a dominant congenital allergic disorder with connective tissue features. LRR, leucine rich repeat domain; PDZ, PSD95-Dlg1-zo-1–like domain; SSID, SMAD–SARA interacting domain. (D) Representative immunoblot of GFP after MYC immunoprecipitation (IP) in 293T cells in which MYC-tagged wild-type STAT3 (MYC- STAT3 WT ) and GFP-tagged ERBIN-mutant (GFP- ERBB2IP mut ) or wild-type (GFP- ERBB2IP WT ) constructs were overexpressed in the presence or absence of STAT3 activation by IL-6. (E) Effect of wild-type ERBIN ( ERBB2IP WT ) or mutant ERBIN ( ERBB2IP mut ) on TGF-β–induced SMAD-reporter activity. AU, arbitrary units. Data are representative or combined from at least three independent experiments and represented as the mean ± SEM. Paired and unpaired two-tailed Student’s t tests and Mann-Whitney tests were used where appropriate. *, P

    Article Snippet: For immunoprecipitation, after the indicated transfection and/or stimulation, 293T cells were lysed in immunoprecipitation lysis buffer (1× Mini protease inhibitor with EDTA [Roche], 20 mM Tris-HCl, pH 7.75, 140 mM NaCl, 1% Triton X-100, and 1 mM PMSF protease inhibitor cocktail [Thermo Fisher Scientific]), precleared, and immunoprecipitated using Protein A/G Plus agarose (Thermo Fisher Scientific) and monoclonal antibodies for SMAD2/3 or by using precoated sepharose beads for STAT3 and MYC (Cell Signaling Technology).

    Techniques: Expressing, Mutagenesis, Binding Assay, Immunoprecipitation, Construct, Activation Assay, Activity Assay, Two Tailed Test, MANN-WHITNEY

    STAT3 negatively regulates TGF-β pathway activation via ERBIN. (A) TGF-β–induced SMAD pathway activation in 293T SMAD-reporter line after short-term culture in the presence of STAT3-activating cytokine IL-6 or IL-11. (B) TGF-β–mediated pathway activation after stimulation in a SMAD-reporter cell line treated with IL-6 (top) and corresponding immunoblot of ERBIN expression from each culture (bottom). (C) TGF-β–induced SMAD-reporter activity after ERBIN knockdown ( siERBB2IP ) or transfection with scrambled siRNA ( siControl ) after 3-d culture in the presence or absence of IL-6. (D) Representative immunoblot of ERBIN, STAT3, and/or SMAD2/3 after immunoprecipitation (IP) of STAT3 or SMAD2/3 as indicated in 293T cells after siRNA-mediated knockdown of ERBIN in the presence or absence of STAT3 activation induced by treatment with IL-6. (E) TGF-β–induced SMAD-reporter activity after either STAT3 knockdown ( siSTAT3 ) or scrambled siRNA transfection and cotransfection with either wild-type ERBIN ( ERBB2IP WT ) or empty vector (EV). (F) TGF-β–induced activation in SMAD-reporter line after transfection with empty vector, wild-type STAT3 ( STAT3 WT ), or dominant-negative STAT3 -mutant ( STAT3 mut ) constructs. (G) TGF-β–induced SMAD-reporter activity after siRNA-mediated STAT3 silencing relative to activity after scrambled siRNA transfection. (H) ERBB2IP mRNA expression in purified control pan-T cells after treatment with 50 ng/ml IL-6. (I) Representative immunoblot of GFP after MYC immunoprecipitation in 293T cells in which MYC-tagged wild-type STAT3 (MYC- STAT3 WT ) or dominant-negative STAT3 (MYC- STAT3 mut ) constructs and GFP-tagged ERBIN wild-type (GFP- ERBB2IP WT ) constructs were overexpressed in the presence or absence of STAT3 activation by IL-6. Data are representative or combined from at least three independent experiments and shown as the mean ± SEM. Unpaired two-tailed Student’s t tests and Wilcoxon matched pairs were used where appropriate. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: ERBIN deficiency links STAT3 and TGF-β pathway defects with atopy in humans

    doi: 10.1084/jem.20161435

    Figure Lengend Snippet: STAT3 negatively regulates TGF-β pathway activation via ERBIN. (A) TGF-β–induced SMAD pathway activation in 293T SMAD-reporter line after short-term culture in the presence of STAT3-activating cytokine IL-6 or IL-11. (B) TGF-β–mediated pathway activation after stimulation in a SMAD-reporter cell line treated with IL-6 (top) and corresponding immunoblot of ERBIN expression from each culture (bottom). (C) TGF-β–induced SMAD-reporter activity after ERBIN knockdown ( siERBB2IP ) or transfection with scrambled siRNA ( siControl ) after 3-d culture in the presence or absence of IL-6. (D) Representative immunoblot of ERBIN, STAT3, and/or SMAD2/3 after immunoprecipitation (IP) of STAT3 or SMAD2/3 as indicated in 293T cells after siRNA-mediated knockdown of ERBIN in the presence or absence of STAT3 activation induced by treatment with IL-6. (E) TGF-β–induced SMAD-reporter activity after either STAT3 knockdown ( siSTAT3 ) or scrambled siRNA transfection and cotransfection with either wild-type ERBIN ( ERBB2IP WT ) or empty vector (EV). (F) TGF-β–induced activation in SMAD-reporter line after transfection with empty vector, wild-type STAT3 ( STAT3 WT ), or dominant-negative STAT3 -mutant ( STAT3 mut ) constructs. (G) TGF-β–induced SMAD-reporter activity after siRNA-mediated STAT3 silencing relative to activity after scrambled siRNA transfection. (H) ERBB2IP mRNA expression in purified control pan-T cells after treatment with 50 ng/ml IL-6. (I) Representative immunoblot of GFP after MYC immunoprecipitation in 293T cells in which MYC-tagged wild-type STAT3 (MYC- STAT3 WT ) or dominant-negative STAT3 (MYC- STAT3 mut ) constructs and GFP-tagged ERBIN wild-type (GFP- ERBB2IP WT ) constructs were overexpressed in the presence or absence of STAT3 activation by IL-6. Data are representative or combined from at least three independent experiments and shown as the mean ± SEM. Unpaired two-tailed Student’s t tests and Wilcoxon matched pairs were used where appropriate. *, P

    Article Snippet: For immunoprecipitation, after the indicated transfection and/or stimulation, 293T cells were lysed in immunoprecipitation lysis buffer (1× Mini protease inhibitor with EDTA [Roche], 20 mM Tris-HCl, pH 7.75, 140 mM NaCl, 1% Triton X-100, and 1 mM PMSF protease inhibitor cocktail [Thermo Fisher Scientific]), precleared, and immunoprecipitated using Protein A/G Plus agarose (Thermo Fisher Scientific) and monoclonal antibodies for SMAD2/3 or by using precoated sepharose beads for STAT3 and MYC (Cell Signaling Technology).

    Techniques: Activation Assay, Expressing, Activity Assay, Transfection, Immunoprecipitation, Cotransfection, Plasmid Preparation, Dominant Negative Mutation, Mutagenesis, Construct, Purification, Two Tailed Test

    Functional characterization of modulation of LRRK2 at T1410. (A) A LanthaScreen assay was used to assess LRRK2 T1410A kinase activity. Titration of wildtype, G2019S, and T1410A (G2019S) LRRK2 proteins demonstrate that mutation T1410A does not affect phosphorylation of LRRKtide. (B) To investigate dimer formation HEK-293T cells were transfected with the indicated LRRK2 constructs. Cell pellets were split equally for lysis by freeze/thaw cycles directly in PBS or lysis with 1% SDS and PBS with sonication, and 10 ug of protein lysate was loaded onto a native gel (3–12% Bis-Tris) or an SDS gel (7% Tris acetate-SDS), respectively. LRRK2 complexes were visualized with the anti-HA antibody by Western blot with standard ECL. Dimer-sized structures are evident (signal from 480 to 550 kDa), monomeric LRRK2 (278 kDa) is only visible by overexposure (Supplemental Figure S1 , [25] ).Western blots representative of five independent experiments are shown. Normalization of the LRRK2 dimer to total LRRK2 protein (SDS-solubilized) was done using densitometry analysis. ** p

    Journal: PLoS ONE

    Article Title: Identification and Characterization of a Leucine-Rich Repeat Kinase 2 (LRRK2) Consensus Phosphorylation Motif

    doi: 10.1371/journal.pone.0013672

    Figure Lengend Snippet: Functional characterization of modulation of LRRK2 at T1410. (A) A LanthaScreen assay was used to assess LRRK2 T1410A kinase activity. Titration of wildtype, G2019S, and T1410A (G2019S) LRRK2 proteins demonstrate that mutation T1410A does not affect phosphorylation of LRRKtide. (B) To investigate dimer formation HEK-293T cells were transfected with the indicated LRRK2 constructs. Cell pellets were split equally for lysis by freeze/thaw cycles directly in PBS or lysis with 1% SDS and PBS with sonication, and 10 ug of protein lysate was loaded onto a native gel (3–12% Bis-Tris) or an SDS gel (7% Tris acetate-SDS), respectively. LRRK2 complexes were visualized with the anti-HA antibody by Western blot with standard ECL. Dimer-sized structures are evident (signal from 480 to 550 kDa), monomeric LRRK2 (278 kDa) is only visible by overexposure (Supplemental Figure S1 , [25] ).Western blots representative of five independent experiments are shown. Normalization of the LRRK2 dimer to total LRRK2 protein (SDS-solubilized) was done using densitometry analysis. ** p

    Article Snippet: Cell pellets were resuspended with either native lysis buffer (phosphate-buffered saline (PBS), pH 7.4, 1x protease and phosphatase inhibitors (Roche Applied Science), lysed by four cycles of freezing and thawing, and analyzed on a 3–12% bis-tris Blue native polyacrylamide gel (Invitrogen) or lysates combined with SDS-lysis buffer (PBS, pH 7.4, 1% SDS, 1xprotease and phosphatase inhibitors (Roche Applied Science) and sonicated with 10% power for 10 s on a Branson dismembranator and analyzed on a 7% Tris acetate SDS gel (Invitrogen).

    Techniques: Functional Assay, Activity Assay, Titration, Mutagenesis, Transfection, Construct, Lysis, Sonication, SDS-Gel, Western Blot

    Western blot analysis of transcription factors present in whole cell lysates collected from primary human foreskin keratinocytes (HFKs) and HPV 31 cells grown in monolayer (0 hour) or suspended in methylcellulose for 7, 16, 24, and 40 hours. Cells pellets were resuspended in RIPA lysis buffer and analyzed on SDS-PAGE gels at examined by western analysis. Antibodies against Sp1, YY1, C/EBP-β, C/EBP-α, E2F2, Oct-1, and c-Jun were used to probe the membrane. GAPDH was used as a loading control.

    Journal: Virology

    Article Title: Regulation of human papillomavirus type 31 gene expression during the differentiation-dependent life cycle through histone modifications and transcription factor binding

    doi: 10.1016/j.virol.2007.12.011

    Figure Lengend Snippet: Western blot analysis of transcription factors present in whole cell lysates collected from primary human foreskin keratinocytes (HFKs) and HPV 31 cells grown in monolayer (0 hour) or suspended in methylcellulose for 7, 16, 24, and 40 hours. Cells pellets were resuspended in RIPA lysis buffer and analyzed on SDS-PAGE gels at examined by western analysis. Antibodies against Sp1, YY1, C/EBP-β, C/EBP-α, E2F2, Oct-1, and c-Jun were used to probe the membrane. GAPDH was used as a loading control.

    Article Snippet: Cells were then resuspended in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1) in the presence of Complete Mini protease inhibitors (Roche Diagnostics, NJ, USA) and incubated on ice for ten minutes.

    Techniques: Western Blot, Lysis, SDS Page