pmsf  (Millipore)

 
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    Name:
    PMSF
    Description:

    Catalog Number:
    pmsf-ro
    Price:
    None
    Applications:
    PMSF (C7H7O2SF) inhibits serine proteases such as chymotrypsin, trypsin, thrombin, and the cysteine protease papain (reversible by DTT treatment). It does not inhibit metalloproteases, most cysteine proteases, or aspartic proteases.PMSF (phenylmethylsulfonyl fluoride) has been used for the preparation of protein extracts from tissues and cells prior to Western blotting.
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    Structured Review

    Millipore pmsf
    PMSF

    https://www.bioz.com/result/pmsf/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pmsf - by Bioz Stars, 2021-03
    99/100 stars

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    Related Articles

    Incubation:

    Article Title: Bacillus sp. JR3 esterase LipJ: A new mesophilic enzyme showing traces of a thermophilic past
    Article Snippet: Residual activity was measured at 30°C and pH 7 using a conventional assay on p NP-butyrate, and expressed as a percentage of activity without ions or inhibitors, respectively. .. Supernatant samples of Bacillus sp. JR3 were also incubated in the presence of 1 mM PMSF (phenylmethylsulfonyl fluoride, Sigma) to measure the effect of this serine inhibitor on enzyme activity. .. Kinetic parameters (Vmax and Km) were determined under optimal assay conditions by fitting hyperbolic Michaelis-Menten curves with GraphPad Prism ® software version 6.

    Article Title: A Novel Pathway for Mammary Epithelial Cell Invasion Induced by the Helix-Loop-Helix Protein Id-1
    Article Snippet: Briefly, gels consisted of 8 to 10% polyacrylamide and 3 mg of α-casein or gelatin (Sigma)/ml. .. Concentrated conditioned medium was mixed with nonreducing Laemmli sample buffer and incubated at 37°C for 15 min. After electrophoresis, the gels were incubated for 1 h in 2.5% Triton X-100 at room temperature, followed by 24 to 48 h in substrate buffer (100 mM Tris-HCl [pH 7.4]–15 mM CaCl2 ) in the absence or presence of GM6001 (0.2 mM in DMSO; supplied by Glycomed Corporation and obtained from Z. Werb [ ]), EDTA (10 mM), ortho -phenanthroline (1 mM in DMSO; Sigma), PMSF (5 mM), or AEBSF (0.5 mM; Calbiochem). .. The gels were stained with Coomassie blue for 30 min and were destained with 30% methanol–10% acetic acid.

    Activity Assay:

    Article Title: Bacillus sp. JR3 esterase LipJ: A new mesophilic enzyme showing traces of a thermophilic past
    Article Snippet: Residual activity was measured at 30°C and pH 7 using a conventional assay on p NP-butyrate, and expressed as a percentage of activity without ions or inhibitors, respectively. .. Supernatant samples of Bacillus sp. JR3 were also incubated in the presence of 1 mM PMSF (phenylmethylsulfonyl fluoride, Sigma) to measure the effect of this serine inhibitor on enzyme activity. .. Kinetic parameters (Vmax and Km) were determined under optimal assay conditions by fitting hyperbolic Michaelis-Menten curves with GraphPad Prism ® software version 6.

    Article Title: Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells
    Article Snippet: The reaction mix was incubated at 37°C; and at various time points, aliquots were removed and resolved in a 12.5% SDS-PAGE gel for carbohydrate (CHO) release. .. Intracellular inhibition of protease digestion In order to inhibit intracellular protease activity, 5 × 106 cells/sample were treated with: Complete™ protease inhibitor tablets; either 1× (1 tablet for every 50 mL of media) or 2× (2 tablets for every 50 mL of media); or treated with antipain (2.0 μg/mL) plus leupeptin (2.0 μg/mL) (both from Sigma-Aldrich, St Louis, MO, USA) for cysteine protease inhibition; or aprotinin (2.0 μg/mL) plus PMSF (100 μg/mL) (both from Sigma-Aldrich) for serine protease inhibition; for to 24 hrs. ..

    Lysis:

    Article Title: Main constraints for RNAi induced by expressed long dsRNA in mouse cells
    Article Snippet: Western blotting 3T3 cells were grown in six-well plates. .. Before collection, the cells were washed with PBS and lysed in lysis buffer (20 mM Hepes [pH 7.8], 100 mM NaCl, 1 mM EDTA [pH 8.0], 0.5% IGEPAL-25%, 1 mM fresh DTT, 0.5 mM PMSF, 1 mM NaF, 0.2 mM Na3 VO4 , supplemented with 2× protease inhibitor cocktail set [Millipore], 2× phosphatase inhibitor cocktail set [Millipore], and RiboLock RNase inhibitor [Thermo Fisher Scientific]). .. Proteins were separated on 10% polyacrylamide gel and transferred to a PVFD membrane (Millipore).

    Protease Inhibitor:

    Article Title: Main constraints for RNAi induced by expressed long dsRNA in mouse cells
    Article Snippet: Western blotting 3T3 cells were grown in six-well plates. .. Before collection, the cells were washed with PBS and lysed in lysis buffer (20 mM Hepes [pH 7.8], 100 mM NaCl, 1 mM EDTA [pH 8.0], 0.5% IGEPAL-25%, 1 mM fresh DTT, 0.5 mM PMSF, 1 mM NaF, 0.2 mM Na3 VO4 , supplemented with 2× protease inhibitor cocktail set [Millipore], 2× phosphatase inhibitor cocktail set [Millipore], and RiboLock RNase inhibitor [Thermo Fisher Scientific]). .. Proteins were separated on 10% polyacrylamide gel and transferred to a PVFD membrane (Millipore).

    Article Title: Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells
    Article Snippet: The reaction mix was incubated at 37°C; and at various time points, aliquots were removed and resolved in a 12.5% SDS-PAGE gel for carbohydrate (CHO) release. .. Intracellular inhibition of protease digestion In order to inhibit intracellular protease activity, 5 × 106 cells/sample were treated with: Complete™ protease inhibitor tablets; either 1× (1 tablet for every 50 mL of media) or 2× (2 tablets for every 50 mL of media); or treated with antipain (2.0 μg/mL) plus leupeptin (2.0 μg/mL) (both from Sigma-Aldrich, St Louis, MO, USA) for cysteine protease inhibition; or aprotinin (2.0 μg/mL) plus PMSF (100 μg/mL) (both from Sigma-Aldrich) for serine protease inhibition; for to 24 hrs. ..

    Electrophoresis:

    Article Title: A Novel Pathway for Mammary Epithelial Cell Invasion Induced by the Helix-Loop-Helix Protein Id-1
    Article Snippet: Briefly, gels consisted of 8 to 10% polyacrylamide and 3 mg of α-casein or gelatin (Sigma)/ml. .. Concentrated conditioned medium was mixed with nonreducing Laemmli sample buffer and incubated at 37°C for 15 min. After electrophoresis, the gels were incubated for 1 h in 2.5% Triton X-100 at room temperature, followed by 24 to 48 h in substrate buffer (100 mM Tris-HCl [pH 7.4]–15 mM CaCl2 ) in the absence or presence of GM6001 (0.2 mM in DMSO; supplied by Glycomed Corporation and obtained from Z. Werb [ ]), EDTA (10 mM), ortho -phenanthroline (1 mM in DMSO; Sigma), PMSF (5 mM), or AEBSF (0.5 mM; Calbiochem). .. The gels were stained with Coomassie blue for 30 min and were destained with 30% methanol–10% acetic acid.

    Inhibition:

    Article Title: Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells
    Article Snippet: The reaction mix was incubated at 37°C; and at various time points, aliquots were removed and resolved in a 12.5% SDS-PAGE gel for carbohydrate (CHO) release. .. Intracellular inhibition of protease digestion In order to inhibit intracellular protease activity, 5 × 106 cells/sample were treated with: Complete™ protease inhibitor tablets; either 1× (1 tablet for every 50 mL of media) or 2× (2 tablets for every 50 mL of media); or treated with antipain (2.0 μg/mL) plus leupeptin (2.0 μg/mL) (both from Sigma-Aldrich, St Louis, MO, USA) for cysteine protease inhibition; or aprotinin (2.0 μg/mL) plus PMSF (100 μg/mL) (both from Sigma-Aldrich) for serine protease inhibition; for to 24 hrs. ..

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  • 97
    Millipore pmsf
    Expression of a 120-kDa gelatinase by Id-1-expressing mammary epithelial cells. (A) Gelatin zymogram of conditioned media from SCp2–Id-1A (lane 1) and SCp2–Id-1E (lane 2) cells. Cells were cultured in serum-free medium, and conditioned media were harvested and analyzed on a gelatin substrate gel, as described in Materials and Methods. (B) Gelatin zymogram of conditioned media from SCp2–Id-1A cells either growth arrested by serum deprivation (lane 1) or growing in 5% serum (lane 2). (C) Gelatin zymogram of conditioned media from control SCp2 cells (lane 1) and an uncloned SCp2–Id-1-transfected pooled population (lane 2). Cells were cultured in serum-free medium. (D) Gelatin zymogram of SCp2–Id-1A (lanes 1, 3, and 5) and SCp2–Id-1E (lanes 2, 4, and 6) cell-conditioned media incubated with <t>DMSO</t> (lanes 1 and 2), the MMP inhibitor GM6001 (0.2 mM in DMSO) (lanes 3 and 4), or the serine proteinase inhibitor <t>PMSF</t> (5 mM in DMSO) (lanes 5 and 6). (E) Casein zymogram of conditioned media from SCp2–Id-1A (lanes 1 and 3) and SCp2–Id-1E (lanes 2 and 4) cells incubated with DMSO (lanes 1 and 2) or GM6001 (lanes 3 and 4). In panels A through D, arrows mark the positions of the 120-kDa MMP.
    Pmsf, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pmsf/product/Millipore
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    Expression of a 120-kDa gelatinase by Id-1-expressing mammary epithelial cells. (A) Gelatin zymogram of conditioned media from SCp2–Id-1A (lane 1) and SCp2–Id-1E (lane 2) cells. Cells were cultured in serum-free medium, and conditioned media were harvested and analyzed on a gelatin substrate gel, as described in Materials and Methods. (B) Gelatin zymogram of conditioned media from SCp2–Id-1A cells either growth arrested by serum deprivation (lane 1) or growing in 5% serum (lane 2). (C) Gelatin zymogram of conditioned media from control SCp2 cells (lane 1) and an uncloned SCp2–Id-1-transfected pooled population (lane 2). Cells were cultured in serum-free medium. (D) Gelatin zymogram of SCp2–Id-1A (lanes 1, 3, and 5) and SCp2–Id-1E (lanes 2, 4, and 6) cell-conditioned media incubated with DMSO (lanes 1 and 2), the MMP inhibitor GM6001 (0.2 mM in DMSO) (lanes 3 and 4), or the serine proteinase inhibitor PMSF (5 mM in DMSO) (lanes 5 and 6). (E) Casein zymogram of conditioned media from SCp2–Id-1A (lanes 1 and 3) and SCp2–Id-1E (lanes 2 and 4) cells incubated with DMSO (lanes 1 and 2) or GM6001 (lanes 3 and 4). In panels A through D, arrows mark the positions of the 120-kDa MMP.

    Journal: Molecular and Cellular Biology

    Article Title: A Novel Pathway for Mammary Epithelial Cell Invasion Induced by the Helix-Loop-Helix Protein Id-1

    doi:

    Figure Lengend Snippet: Expression of a 120-kDa gelatinase by Id-1-expressing mammary epithelial cells. (A) Gelatin zymogram of conditioned media from SCp2–Id-1A (lane 1) and SCp2–Id-1E (lane 2) cells. Cells were cultured in serum-free medium, and conditioned media were harvested and analyzed on a gelatin substrate gel, as described in Materials and Methods. (B) Gelatin zymogram of conditioned media from SCp2–Id-1A cells either growth arrested by serum deprivation (lane 1) or growing in 5% serum (lane 2). (C) Gelatin zymogram of conditioned media from control SCp2 cells (lane 1) and an uncloned SCp2–Id-1-transfected pooled population (lane 2). Cells were cultured in serum-free medium. (D) Gelatin zymogram of SCp2–Id-1A (lanes 1, 3, and 5) and SCp2–Id-1E (lanes 2, 4, and 6) cell-conditioned media incubated with DMSO (lanes 1 and 2), the MMP inhibitor GM6001 (0.2 mM in DMSO) (lanes 3 and 4), or the serine proteinase inhibitor PMSF (5 mM in DMSO) (lanes 5 and 6). (E) Casein zymogram of conditioned media from SCp2–Id-1A (lanes 1 and 3) and SCp2–Id-1E (lanes 2 and 4) cells incubated with DMSO (lanes 1 and 2) or GM6001 (lanes 3 and 4). In panels A through D, arrows mark the positions of the 120-kDa MMP.

    Article Snippet: Concentrated conditioned medium was mixed with nonreducing Laemmli sample buffer and incubated at 37°C for 15 min. After electrophoresis, the gels were incubated for 1 h in 2.5% Triton X-100 at room temperature, followed by 24 to 48 h in substrate buffer (100 mM Tris-HCl [pH 7.4]–15 mM CaCl2 ) in the absence or presence of GM6001 (0.2 mM in DMSO; supplied by Glycomed Corporation and obtained from Z. Werb [ ]), EDTA (10 mM), ortho -phenanthroline (1 mM in DMSO; Sigma), PMSF (5 mM), or AEBSF (0.5 mM; Calbiochem).

    Techniques: Expressing, Cell Culture, Transfection, Incubation

    The cortactin repeat has a dynamic conformation in solution (A) The endogenous cysteine residues 112 and 246 of the cortactin repeat were crosslinked and the ability of the crosslinked protein to bind F-actin was tested in a co-sedimentation assay (Materials and Methods). The pellet (P) and supernatant (S) were analyzed on 12% SDS-PAGE. Lanes 1 and 2, uncrosslinked cortactin; lanes 3 and 4, disulfide crosslinked cortactin (xl-cortactin); lanes 5 and 6, reduction of the disulfide crosslink with 10mM DTT. (B) The ability of the NEM-labeled cortactin repeat (at residues C112 and C246) to bind to F-actin was assessed using a co-sedimentation assay (Materials and Methods). The pellet and supernatant were analyzed on 12% SDS-PAGE. Lanes 1 and 2, non-labeled cortactin; lanes 3 and 4, NEM-labeled cortactin. (C) Crosslinking of cortactin repeat constructs containing pairs of cysteine residues at positions 83 and 306 (left), 83 and 246 (middle), and 83 and 112 (right). Prior to each crosslinking reaction, DTT was removed from the samples on a Sephadex G-50 spin column equilibrated with 20 mM HEPES pH 7.0, 0.1 M KCl, and 100μM PMSF. The crosslinking reactions were performed on ice and catalyzed by addition of 10μM CuSO 4 . Each cysteine pair was crosslinked either directly by disulfide bond formation (zero length crosslink), or using the crosslinking reagents DBB (4.4 Å span) or MTS-6 (9.6 Å span).

    Journal: Cell motility and the cytoskeleton

    Article Title: The Actin-binding Domain of Cortactin is Dynamic and Unstructured and Affects Lateral and Longitudinal Contacts in F-actin

    doi: 10.1002/cm.20328

    Figure Lengend Snippet: The cortactin repeat has a dynamic conformation in solution (A) The endogenous cysteine residues 112 and 246 of the cortactin repeat were crosslinked and the ability of the crosslinked protein to bind F-actin was tested in a co-sedimentation assay (Materials and Methods). The pellet (P) and supernatant (S) were analyzed on 12% SDS-PAGE. Lanes 1 and 2, uncrosslinked cortactin; lanes 3 and 4, disulfide crosslinked cortactin (xl-cortactin); lanes 5 and 6, reduction of the disulfide crosslink with 10mM DTT. (B) The ability of the NEM-labeled cortactin repeat (at residues C112 and C246) to bind to F-actin was assessed using a co-sedimentation assay (Materials and Methods). The pellet and supernatant were analyzed on 12% SDS-PAGE. Lanes 1 and 2, non-labeled cortactin; lanes 3 and 4, NEM-labeled cortactin. (C) Crosslinking of cortactin repeat constructs containing pairs of cysteine residues at positions 83 and 306 (left), 83 and 246 (middle), and 83 and 112 (right). Prior to each crosslinking reaction, DTT was removed from the samples on a Sephadex G-50 spin column equilibrated with 20 mM HEPES pH 7.0, 0.1 M KCl, and 100μM PMSF. The crosslinking reactions were performed on ice and catalyzed by addition of 10μM CuSO 4 . Each cysteine pair was crosslinked either directly by disulfide bond formation (zero length crosslink), or using the crosslinking reagents DBB (4.4 Å span) or MTS-6 (9.6 Å span).

    Article Snippet: Sephadex G-50, N-ethylmaleimide (NEM), ATP, phalloidin, DTT and PMSF were purchased from Sigma Chemical Company (St. Louis, MO).

    Techniques: Sedimentation, SDS Page, Labeling, Construct

    Shedding of sHLA-E into JEV-infected culture supernatants. Panel A: Western blot for sHLA-E in cell-culture supernatants from uninfected (Con) and infected HBMEC and ECV at various times p.i. as labeled. ‘Gelatinase’ represents enzyme activity in normal (Con) ECV cells that were either treated with IFN-γ or infected with JEV for the indicated times. Panel B: Cell-culture supernatants were collected from ECV or HBMEC cells that were infected for 24 h with JEV and cultured in the presence of 0 µM, 5 µM and 10 µM GM6001. Panel C: Cell-culture supernatants were collected from uninfected (Con) and 24 h JEV-infected (Top) or TNF-α treated (Bottom) ECV cells that were left untreated or treated with 10 µM GM6001, 10 µM leupeptin, 1 mM PMSF, and 100 µM chloroquine. Panel D: Total RNA was isolated from uninfected (Con) and 24 h JEV-infected ECV cells that were left untreated or treated with 10 µM GM6001, 10 µM leupeptin, 1 mM PMSF, and 100 µM chloroquine. RT-PCR was performed for the JEV envelope and 18s RNA controls. All inhibitors used in panels B, C and D were present only during the last 8 h of the 24 h-culture.

    Journal: PLoS ONE

    Article Title: Infection of Human Endothelial Cells by Japanese Encephalitis Virus: Increased Expression and Release of Soluble HLA-E

    doi: 10.1371/journal.pone.0079197

    Figure Lengend Snippet: Shedding of sHLA-E into JEV-infected culture supernatants. Panel A: Western blot for sHLA-E in cell-culture supernatants from uninfected (Con) and infected HBMEC and ECV at various times p.i. as labeled. ‘Gelatinase’ represents enzyme activity in normal (Con) ECV cells that were either treated with IFN-γ or infected with JEV for the indicated times. Panel B: Cell-culture supernatants were collected from ECV or HBMEC cells that were infected for 24 h with JEV and cultured in the presence of 0 µM, 5 µM and 10 µM GM6001. Panel C: Cell-culture supernatants were collected from uninfected (Con) and 24 h JEV-infected (Top) or TNF-α treated (Bottom) ECV cells that were left untreated or treated with 10 µM GM6001, 10 µM leupeptin, 1 mM PMSF, and 100 µM chloroquine. Panel D: Total RNA was isolated from uninfected (Con) and 24 h JEV-infected ECV cells that were left untreated or treated with 10 µM GM6001, 10 µM leupeptin, 1 mM PMSF, and 100 µM chloroquine. RT-PCR was performed for the JEV envelope and 18s RNA controls. All inhibitors used in panels B, C and D were present only during the last 8 h of the 24 h-culture.

    Article Snippet: Galardin (GM6001), a broad spectrum MMP inhibitor, was obtained from Merck, USA while leupeptin and PMSF were obtained from Sigma-aldrich, India.

    Techniques: Infection, Western Blot, Cell Culture, Labeling, Activity Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

    Trypsin digestion generates a 69-kDa Ku86 protein from a full-length cloned human MM Ku86 protein (rhKu86) in vitro . Full-length rhKu86 was first expressed and purified from COS cells. Serial digestion (A) of rhKu86 (6.5 μg/sample) by trypsin (0.065 μg of Trypsin Gold/reaction up to 20 mins) was then performed using a limited proteolysis protocol from the manufacturer. The reactions were stopped by rapid cooling on ice; and the products of trypsin digestion were resolved by SDS PAGE and immunoblotted S10B1 anti-Ku86 mAb. The effect of protease inhibitors aprotinin (2.0 μg/mL final concentration) plus PMSF (100 μg/mL final concentration) on trypsin digestion was also determined on a larger sample of rhKu86 (13.0 μg/sample). Trypsin digestion (0.065 μg of Trypsin Gold/reaction for 1 min) of rhKu86 was performed as above either without (lane 1) or with (lane 2) protease inhibitors (B).

    Journal: Cancer Cell International

    Article Title: Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells

    doi: 10.1186/1475-2867-8-4

    Figure Lengend Snippet: Trypsin digestion generates a 69-kDa Ku86 protein from a full-length cloned human MM Ku86 protein (rhKu86) in vitro . Full-length rhKu86 was first expressed and purified from COS cells. Serial digestion (A) of rhKu86 (6.5 μg/sample) by trypsin (0.065 μg of Trypsin Gold/reaction up to 20 mins) was then performed using a limited proteolysis protocol from the manufacturer. The reactions were stopped by rapid cooling on ice; and the products of trypsin digestion were resolved by SDS PAGE and immunoblotted S10B1 anti-Ku86 mAb. The effect of protease inhibitors aprotinin (2.0 μg/mL final concentration) plus PMSF (100 μg/mL final concentration) on trypsin digestion was also determined on a larger sample of rhKu86 (13.0 μg/sample). Trypsin digestion (0.065 μg of Trypsin Gold/reaction for 1 min) of rhKu86 was performed as above either without (lane 1) or with (lane 2) protease inhibitors (B).

    Article Snippet: Intracellular inhibition of protease digestion In order to inhibit intracellular protease activity, 5 × 106 cells/sample were treated with: Complete™ protease inhibitor tablets; either 1× (1 tablet for every 50 mL of media) or 2× (2 tablets for every 50 mL of media); or treated with antipain (2.0 μg/mL) plus leupeptin (2.0 μg/mL) (both from Sigma-Aldrich, St Louis, MO, USA) for cysteine protease inhibition; or aprotinin (2.0 μg/mL) plus PMSF (100 μg/mL) (both from Sigma-Aldrich) for serine protease inhibition; for to 24 hrs.

    Techniques: Clone Assay, In Vitro, Purification, SDS Page, Concentration Assay

    Proteolysis plays an important role in the intracellular generation of the 69-kDa Ku86v variant protein in MM cell lines . RPMI 8226 (A) and SGH-MM5 (data not shown) MM cell lines (5.0 × 10 6 cells/sample) were first incubated for 18 hrs with either 1× or 2× Complete™ protease inhibitor tablets (lanes 2 and 3); and then washed and checked for viability using trypan blue exclusion assay. Mock experiments in which cell lines were incubated for 18 hrs with media alone (lane 1) served as negative controls. Cell lysates were resolved by SDS-PAGE and immunoblotted using S10B1 anti-Ku86 mAb. The RPMI 8226 MM cell line s (B) was also incubated for 24 hrs with 2× Complete™ protease inhibitor tablets (lane 2), antipain (2.0 μg/mL final concentration) plus leupeptin (2.0 μg/mL final concentration) (lane 3), or aprotinin (2.0 μg/mL final concentration) plus PMSF (100 μg/mL final concentration) (lane 4) and analyzed in the same way as above. Mock experiments in which cell lines were incubated for 24 hrs with media alone (lane 1) again served as negative controls. Membranes were stripped and re-probed using anti-actin mAb (control) to confirm equal protein loading. Cell lysates were resolved by SDS-PAGE and immunoblotted using S10B1 anti-Ku86 mAb. Relative expression of 69-kDa Ku86v-DNA complexes (normalized to weakest band) was determined using image densitometry. All experiments were performed in triplicate.

    Journal: Cancer Cell International

    Article Title: Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells

    doi: 10.1186/1475-2867-8-4

    Figure Lengend Snippet: Proteolysis plays an important role in the intracellular generation of the 69-kDa Ku86v variant protein in MM cell lines . RPMI 8226 (A) and SGH-MM5 (data not shown) MM cell lines (5.0 × 10 6 cells/sample) were first incubated for 18 hrs with either 1× or 2× Complete™ protease inhibitor tablets (lanes 2 and 3); and then washed and checked for viability using trypan blue exclusion assay. Mock experiments in which cell lines were incubated for 18 hrs with media alone (lane 1) served as negative controls. Cell lysates were resolved by SDS-PAGE and immunoblotted using S10B1 anti-Ku86 mAb. The RPMI 8226 MM cell line s (B) was also incubated for 24 hrs with 2× Complete™ protease inhibitor tablets (lane 2), antipain (2.0 μg/mL final concentration) plus leupeptin (2.0 μg/mL final concentration) (lane 3), or aprotinin (2.0 μg/mL final concentration) plus PMSF (100 μg/mL final concentration) (lane 4) and analyzed in the same way as above. Mock experiments in which cell lines were incubated for 24 hrs with media alone (lane 1) again served as negative controls. Membranes were stripped and re-probed using anti-actin mAb (control) to confirm equal protein loading. Cell lysates were resolved by SDS-PAGE and immunoblotted using S10B1 anti-Ku86 mAb. Relative expression of 69-kDa Ku86v-DNA complexes (normalized to weakest band) was determined using image densitometry. All experiments were performed in triplicate.

    Article Snippet: Intracellular inhibition of protease digestion In order to inhibit intracellular protease activity, 5 × 106 cells/sample were treated with: Complete™ protease inhibitor tablets; either 1× (1 tablet for every 50 mL of media) or 2× (2 tablets for every 50 mL of media); or treated with antipain (2.0 μg/mL) plus leupeptin (2.0 μg/mL) (both from Sigma-Aldrich, St Louis, MO, USA) for cysteine protease inhibition; or aprotinin (2.0 μg/mL) plus PMSF (100 μg/mL) (both from Sigma-Aldrich) for serine protease inhibition; for to 24 hrs.

    Techniques: Variant Assay, Incubation, Protease Inhibitor, Trypan Blue Exclusion Assay, SDS Page, Concentration Assay, Expressing