mg 132  (Millipore)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    Name:
    MG 132
    Description:

    Catalog Number:
    SML1135
    Price:
    None
    Buy from Supplier


    Structured Review

    Millipore mg 132
    MG 132

    https://www.bioz.com/result/mg 132/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mg 132 - by Bioz Stars, 2021-09
    86/100 stars

    Images

    1) Product Images from "Praja1 RING‐finger E3 ubiquitin ligase suppresses neuronal cytoplasmic TDP‐43 aggregate formation"

    Article Title: Praja1 RING‐finger E3 ubiquitin ligase suppresses neuronal cytoplasmic TDP‐43 aggregate formation

    Journal: Neuropathology

    doi: 10.1111/neup.12694

    Suppression of phosphorylation and aggregate formation of TDP‐43 by PJA1 via proteasomal degradation. (A) Western blot analysis of suppressive effects of adenoviral PJA1 on phosphorylation and aggregate formation of TDP‐43 after wild‐type (WT) and CTF TDP‐43 adenovirus infection in the absence or presence of proteasome inhibitors MG‐132 (0.5 μM), lactacystin (1 μM), epoxomicin (0.1 μM), and bortezomib (0.5 μM). These proteasome inhibitors equally enhance formation of RIPA‐insoluble phosphorylated TDP‐43, which was suppressed by adenoviral PJA1. (B) Western blot analysis of suppressive effects of adenoviral HSF1 and PJA1 on phosphorylation and aggregate formation of TDP‐43 after WT and CTF TDP‐43 adenovirus infection in the absence or presence of proteasome inhibitor MG‐132 (0.5 μM) and autophagy inhibitors 3‐methyladenine (5 mM), bafilomycin A1 (0.1 μM), and wortmannin (1 μM). These autophagy inhibitors give rise to no significant effects on phosphorylation and aggregate formation of TDP‐43 (lanes 3–8) nor antagonizing effects on HSF1 or PJA1 (lanes 10–16). (C) Western blot analysis of adenoviruses expressing HSF1 lacking its catalytic domain (HSF1∆ [380–529]) and PJA1∆R, both of which are defective of suppressive effects on TDP‐43 phosphorylation and aggregation (lanes 3, 6). Adenoviral HSF1∆, acting as a dominant‐negative form, inhibits suppressing effects of WT HSF1 on TDP‐43 aggregate formation (lane 1–4), while PJA1∆R does not act in a dominant‐negative manner to suppress WT PJA1 or HSF1 function (lane 5–8).
    Figure Legend Snippet: Suppression of phosphorylation and aggregate formation of TDP‐43 by PJA1 via proteasomal degradation. (A) Western blot analysis of suppressive effects of adenoviral PJA1 on phosphorylation and aggregate formation of TDP‐43 after wild‐type (WT) and CTF TDP‐43 adenovirus infection in the absence or presence of proteasome inhibitors MG‐132 (0.5 μM), lactacystin (1 μM), epoxomicin (0.1 μM), and bortezomib (0.5 μM). These proteasome inhibitors equally enhance formation of RIPA‐insoluble phosphorylated TDP‐43, which was suppressed by adenoviral PJA1. (B) Western blot analysis of suppressive effects of adenoviral HSF1 and PJA1 on phosphorylation and aggregate formation of TDP‐43 after WT and CTF TDP‐43 adenovirus infection in the absence or presence of proteasome inhibitor MG‐132 (0.5 μM) and autophagy inhibitors 3‐methyladenine (5 mM), bafilomycin A1 (0.1 μM), and wortmannin (1 μM). These autophagy inhibitors give rise to no significant effects on phosphorylation and aggregate formation of TDP‐43 (lanes 3–8) nor antagonizing effects on HSF1 or PJA1 (lanes 10–16). (C) Western blot analysis of adenoviruses expressing HSF1 lacking its catalytic domain (HSF1∆ [380–529]) and PJA1∆R, both of which are defective of suppressive effects on TDP‐43 phosphorylation and aggregation (lanes 3, 6). Adenoviral HSF1∆, acting as a dominant‐negative form, inhibits suppressing effects of WT HSF1 on TDP‐43 aggregate formation (lane 1–4), while PJA1∆R does not act in a dominant‐negative manner to suppress WT PJA1 or HSF1 function (lane 5–8).

    Techniques Used: Western Blot, Infection, Expressing, Dominant Negative Mutation

    Suppression of phosphorylation and aggregate formation of TDP‐43 by PJA1. (A) Schematic presentation of the primary structure of rat, mouse, and human PJA1. RING‐H2 domains located in the C‐terminal region are depicted with dark gray boxes. h, human; m, mouse; N/I, not identified; r, rat. (B) Western blot analysis of endogenous PJA1 induction by the HSF1 adenovirus in 1464R cells. The 1464R cells are infected with TDP‐43 and HSF1 adenoviruses as indicated in Figure 1A . PJA1 expression is enhanced by adenoviral HSF1 and 0.5 μM MG‐132 treatments, predominantly in its shortest form (variant X4; asterisk approximately 50 kD). (C) Western blot analysis of EGFP‐tagged adenoviruses expressing rat variant X4 (AxrPJA1x4EGFP), rat variant X1 (AxrPJA1x1EGFP), rat RING‐H2‐lacking variant X1 (AxrPJA1ΔREGFP), human variant V1 (AxhPJA1v1EGFP), and human RING‐H2‐lacking variant V1 (AxhPJA1ΔREGFP). Increased expression of wild‐type (WT) PJA1 in the presence of 0.5 μM MG‐132 is due to inhibition of proteasomal degradation of self‐ubiquitinated PJA1, while expression of PJA1 lacking RING‐H2 domain is independent of proteasomal activity. (D) EGFP fluorescence of 1464R‐derived neurons infected with PJA1EGFP adenoviruses in the absence or presence of MG‐132. The nucleus is counterstained with Hoechst 33342. WT PJA1EGFP fluorescence is enhanced in the presence of MG‐132 due to proteasomal inhibition, while EGFP expression of PJA1 lacking RING‐H2 domain is not affected by MG‐132 treatment. (E) Fluorescence microscopy of 1464R‐derived neurons infected with adenoviruses expressing DsRed‐tagged human WT and CTF TDP‐43 and EGFP‐tagged PJA1 constructs in the presence of MG‐132. Adenoviruses expressing rat and human WT PJA1 (AxrPJA1x4EGFP, AxrPJA1x1EGFP, AxhPJA1v1EGFP) suppress DsRed‐positive cytoplasmic TDP‐43 aggregate formation, but those lacking RING‐H2 domain (AxrPJA1ΔREGFP, AxhPJA1ΔREGFP) do not. (F) Western blot analysis of 1464R‐derived neurons infected with adenoviruses expressing DsRed‐tagged human WT and CTF TDP‐43 and EGFP‐tagged HSF1 and PJA1 constructs in the absence or presence of 0.5 μM MG‐132. Adenoviruses expressing rat and human HSF1 and WT PJA1 (AxrPJA1x4EGFP, AxrPJA1x1EGFP, AxhPJA1v1EGFP) suppress formation of RIPA‐insoluble phosphorylated TDP‐43, but those lacking RING‐H2 domain (AxrPJA1ΔREGFP, AxhPJA1ΔREGFP) do not. (G) Cell viability assay using CCK‐8 reagent. The y ‐axis depicts cell survival ratio relative to untreated control. There was no significant difference in cell survival between EGFP, hHSF1EGFP, and hPJA1v1EGFP adenovirus infections followed by MG‐132 treatment, while both longer (48 versus 24 h) incubation and higher (0, 0.5, 1, versus 2 μM) concentration of MG‐132 exacerbate the cell survival.
    Figure Legend Snippet: Suppression of phosphorylation and aggregate formation of TDP‐43 by PJA1. (A) Schematic presentation of the primary structure of rat, mouse, and human PJA1. RING‐H2 domains located in the C‐terminal region are depicted with dark gray boxes. h, human; m, mouse; N/I, not identified; r, rat. (B) Western blot analysis of endogenous PJA1 induction by the HSF1 adenovirus in 1464R cells. The 1464R cells are infected with TDP‐43 and HSF1 adenoviruses as indicated in Figure 1A . PJA1 expression is enhanced by adenoviral HSF1 and 0.5 μM MG‐132 treatments, predominantly in its shortest form (variant X4; asterisk approximately 50 kD). (C) Western blot analysis of EGFP‐tagged adenoviruses expressing rat variant X4 (AxrPJA1x4EGFP), rat variant X1 (AxrPJA1x1EGFP), rat RING‐H2‐lacking variant X1 (AxrPJA1ΔREGFP), human variant V1 (AxhPJA1v1EGFP), and human RING‐H2‐lacking variant V1 (AxhPJA1ΔREGFP). Increased expression of wild‐type (WT) PJA1 in the presence of 0.5 μM MG‐132 is due to inhibition of proteasomal degradation of self‐ubiquitinated PJA1, while expression of PJA1 lacking RING‐H2 domain is independent of proteasomal activity. (D) EGFP fluorescence of 1464R‐derived neurons infected with PJA1EGFP adenoviruses in the absence or presence of MG‐132. The nucleus is counterstained with Hoechst 33342. WT PJA1EGFP fluorescence is enhanced in the presence of MG‐132 due to proteasomal inhibition, while EGFP expression of PJA1 lacking RING‐H2 domain is not affected by MG‐132 treatment. (E) Fluorescence microscopy of 1464R‐derived neurons infected with adenoviruses expressing DsRed‐tagged human WT and CTF TDP‐43 and EGFP‐tagged PJA1 constructs in the presence of MG‐132. Adenoviruses expressing rat and human WT PJA1 (AxrPJA1x4EGFP, AxrPJA1x1EGFP, AxhPJA1v1EGFP) suppress DsRed‐positive cytoplasmic TDP‐43 aggregate formation, but those lacking RING‐H2 domain (AxrPJA1ΔREGFP, AxhPJA1ΔREGFP) do not. (F) Western blot analysis of 1464R‐derived neurons infected with adenoviruses expressing DsRed‐tagged human WT and CTF TDP‐43 and EGFP‐tagged HSF1 and PJA1 constructs in the absence or presence of 0.5 μM MG‐132. Adenoviruses expressing rat and human HSF1 and WT PJA1 (AxrPJA1x4EGFP, AxrPJA1x1EGFP, AxhPJA1v1EGFP) suppress formation of RIPA‐insoluble phosphorylated TDP‐43, but those lacking RING‐H2 domain (AxrPJA1ΔREGFP, AxhPJA1ΔREGFP) do not. (G) Cell viability assay using CCK‐8 reagent. The y ‐axis depicts cell survival ratio relative to untreated control. There was no significant difference in cell survival between EGFP, hHSF1EGFP, and hPJA1v1EGFP adenovirus infections followed by MG‐132 treatment, while both longer (48 versus 24 h) incubation and higher (0, 0.5, 1, versus 2 μM) concentration of MG‐132 exacerbate the cell survival.

    Techniques Used: Western Blot, Infection, Expressing, Variant Assay, Inhibition, Activity Assay, Fluorescence, Derivative Assay, Microscopy, Construct, Viability Assay, CCK-8 Assay, Incubation, Concentration Assay

    PJA1 binding to TDP‐43 and E2 ubiquitin conjugating enzyme UBE2E3. (A) Western blot analysis of suppressive effects of adenovirus expressing human PJA1v1, but not PJA1ΔR, on phosphorylation and aggregate formation of TDP‐43 after DsRed‐ and FLAG‐tagged wild‐type (WT) and CTF TDP‐43 adenovirus infection in the presence of 0.5 μM MG‐132 (lanes 3–8). The CTF TDP‐43 adenovirus preferably induces RIPA‐insoluble phosphorylated TDP‐43 (lanes 4, 6). Adenovirally‐induced hPJA1v1 and hPJA1ΔR is consistently localized in both RIPA‐soluble and insoluble fractions (lanes 3, 5, 7, 8), and adenoviral hPJA1v1, but not hPJA1ΔR, decreases RIPA‐insoluble phosphorylated CTF TDP‐43 (lanes 5, 7) in a similar manner to the experiment shown in Figure 3F . Endogenous UBE2E3 is also detected both in RIPA‐soluble and insoluble fractions. (B) The co‐immunoprecipitation (Co‐IP) assay shows that PJA1 preferentially binds to CTF TDP‐43 rather than WT TDP‐43 (PJA1 blot; lanes 3, 5, 7, 8), while CTF TDP‐43 is consistently ubiquitinated irresp ective of adenoviral PJA1 induction (Ubiquitin K48 blot; lanes 4–8). Both native and adenoviral UBE2E3 are co‐immunoprecipitated with WT and CTF TDP‐43 (UBE2E3 and Myc‐Tag blots). (C) Co‐IP assay indicates that TDP‐43 and/or PJA1 bind to UBE2E3 but not UBE2D2/3 or UBE2K. (D) Fluorescence micrographs of TuJ1‐immunoreactive differentiated neurons co‐infected with DsRed‐tagged WT and CTF TDP‐43 adenoviruses and EGFP‐tagged hPJA1v1 (top row) or hPJA1ΔR (bottom row) adenovirus in the presence of 0.5 μM MG‐132. The nucleus is counterstained with Hoechst 33342. Two types of co‐localization (i.e., perinuclear round fluorescence [arrows] and cytoplasmic amorphous aggregates [arrowheads]), are observed.
    Figure Legend Snippet: PJA1 binding to TDP‐43 and E2 ubiquitin conjugating enzyme UBE2E3. (A) Western blot analysis of suppressive effects of adenovirus expressing human PJA1v1, but not PJA1ΔR, on phosphorylation and aggregate formation of TDP‐43 after DsRed‐ and FLAG‐tagged wild‐type (WT) and CTF TDP‐43 adenovirus infection in the presence of 0.5 μM MG‐132 (lanes 3–8). The CTF TDP‐43 adenovirus preferably induces RIPA‐insoluble phosphorylated TDP‐43 (lanes 4, 6). Adenovirally‐induced hPJA1v1 and hPJA1ΔR is consistently localized in both RIPA‐soluble and insoluble fractions (lanes 3, 5, 7, 8), and adenoviral hPJA1v1, but not hPJA1ΔR, decreases RIPA‐insoluble phosphorylated CTF TDP‐43 (lanes 5, 7) in a similar manner to the experiment shown in Figure 3F . Endogenous UBE2E3 is also detected both in RIPA‐soluble and insoluble fractions. (B) The co‐immunoprecipitation (Co‐IP) assay shows that PJA1 preferentially binds to CTF TDP‐43 rather than WT TDP‐43 (PJA1 blot; lanes 3, 5, 7, 8), while CTF TDP‐43 is consistently ubiquitinated irresp ective of adenoviral PJA1 induction (Ubiquitin K48 blot; lanes 4–8). Both native and adenoviral UBE2E3 are co‐immunoprecipitated with WT and CTF TDP‐43 (UBE2E3 and Myc‐Tag blots). (C) Co‐IP assay indicates that TDP‐43 and/or PJA1 bind to UBE2E3 but not UBE2D2/3 or UBE2K. (D) Fluorescence micrographs of TuJ1‐immunoreactive differentiated neurons co‐infected with DsRed‐tagged WT and CTF TDP‐43 adenoviruses and EGFP‐tagged hPJA1v1 (top row) or hPJA1ΔR (bottom row) adenovirus in the presence of 0.5 μM MG‐132. The nucleus is counterstained with Hoechst 33342. Two types of co‐localization (i.e., perinuclear round fluorescence [arrows] and cytoplasmic amorphous aggregates [arrowheads]), are observed.

    Techniques Used: Binding Assay, Western Blot, Expressing, Infection, Co-Immunoprecipitation Assay, Immunoprecipitation, Fluorescence

    Suppression of cytoplasmic TDP‐43 aggregate formation by HSF1. (A) Schematic presentation of 1464R cell culture experiments for adenoviral TDP‐43 aggregate formation. The 1464R cells are cultured in differentiation medium containing all trans retinoic acid (ATRA) and infected with adenoviruses expressing DsRed‐tagged human wild‐type (WT) (AxDsRhWTTDP43) and CTF (AxDsRhCTFTDP43) TDP‐43 and EGFP‐tagged human HSF1 (AxhHSF1EGFP) followed by incubation with 0.5 μM MG‐132. (B) Fluorescence micrographs showing formation of DsRed‐positive dense cytoplasmic aggregates (arrows) in TuJ1‐immunoreactive differentiated neurons induced by TDP‐43 adenoviruses in the presence of 0.5 μM MG‐132 (top row). The TDP‐43 aggregate formation is markedly suppressed by co‐infection of an EGFP‐tagged HSF1 adenovirus (bottom row). Adenoviral HSF1 is localized to nuclei. The nucleus is counterstained with Hoechst 33342. (C) Western blot analysis shows that HSF1 adenovirus infection markedly decreased the amount of adenovirus‐induced RIPA‐insoluble fractions containing phosphorylated TDP‐43.
    Figure Legend Snippet: Suppression of cytoplasmic TDP‐43 aggregate formation by HSF1. (A) Schematic presentation of 1464R cell culture experiments for adenoviral TDP‐43 aggregate formation. The 1464R cells are cultured in differentiation medium containing all trans retinoic acid (ATRA) and infected with adenoviruses expressing DsRed‐tagged human wild‐type (WT) (AxDsRhWTTDP43) and CTF (AxDsRhCTFTDP43) TDP‐43 and EGFP‐tagged human HSF1 (AxhHSF1EGFP) followed by incubation with 0.5 μM MG‐132. (B) Fluorescence micrographs showing formation of DsRed‐positive dense cytoplasmic aggregates (arrows) in TuJ1‐immunoreactive differentiated neurons induced by TDP‐43 adenoviruses in the presence of 0.5 μM MG‐132 (top row). The TDP‐43 aggregate formation is markedly suppressed by co‐infection of an EGFP‐tagged HSF1 adenovirus (bottom row). Adenoviral HSF1 is localized to nuclei. The nucleus is counterstained with Hoechst 33342. (C) Western blot analysis shows that HSF1 adenovirus infection markedly decreased the amount of adenovirus‐induced RIPA‐insoluble fractions containing phosphorylated TDP‐43.

    Techniques Used: Cell Culture, Infection, Expressing, Incubation, Fluorescence, Western Blot

    Screening of candidate genes to suppress TDP‐43 aggregate formation in 1464R‐derived neurons. (A) Schematic presentation of 1464R cell transfection experiments for the screening of candidate genes. The dissociated 1464R cells on coverslips are immediately transfected with plasmids expressing EGFP‐tagged candidate genes followed by TDP‐43 adenovirus infection and MG‐132 incubation. (B) Fluorescence microscopy of fixed cells expressing EGFP‐tagged AMIGO2 (top row), PJA1 (middle row), and HEBP2 (bottom row) that do not contain adenoviral DsRed‐positive TDP‐43 aggregates. (C) Schematic presentation of 1464R cell culture experiments for screening EGFP‐tagged adenoviruses expressing gene of interest (AxGOIEGFPs) to suppress adenoviral TDP‐43 aggregate formation. (D) Western blot analysis of suppressive effects of EGFP‐tagged adenoviruses expressing ACP5, AMIGO2, CREB3L1, PJA1, HEBP2, and DNAJB2a on adenoviral TDP‐43 aggregate formation. (E) Densitometric analysis of the Western blot data of (D) ( n = 3) calibrated with GADPH signals. Data are expressed as relative density compared with AxEGFP‐treated positive control samples. Results are presented as mean ± SD. Statistical comparison is done by two‐tailed unpaired t ‐test (* P
    Figure Legend Snippet: Screening of candidate genes to suppress TDP‐43 aggregate formation in 1464R‐derived neurons. (A) Schematic presentation of 1464R cell transfection experiments for the screening of candidate genes. The dissociated 1464R cells on coverslips are immediately transfected with plasmids expressing EGFP‐tagged candidate genes followed by TDP‐43 adenovirus infection and MG‐132 incubation. (B) Fluorescence microscopy of fixed cells expressing EGFP‐tagged AMIGO2 (top row), PJA1 (middle row), and HEBP2 (bottom row) that do not contain adenoviral DsRed‐positive TDP‐43 aggregates. (C) Schematic presentation of 1464R cell culture experiments for screening EGFP‐tagged adenoviruses expressing gene of interest (AxGOIEGFPs) to suppress adenoviral TDP‐43 aggregate formation. (D) Western blot analysis of suppressive effects of EGFP‐tagged adenoviruses expressing ACP5, AMIGO2, CREB3L1, PJA1, HEBP2, and DNAJB2a on adenoviral TDP‐43 aggregate formation. (E) Densitometric analysis of the Western blot data of (D) ( n = 3) calibrated with GADPH signals. Data are expressed as relative density compared with AxEGFP‐treated positive control samples. Results are presented as mean ± SD. Statistical comparison is done by two‐tailed unpaired t ‐test (* P

    Techniques Used: Derivative Assay, Transfection, Expressing, Infection, Incubation, Fluorescence, Microscopy, Cell Culture, Western Blot, Positive Control, Two Tailed Test

    2) Product Images from "Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells"

    Article Title: Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells

    Journal: Oncology Research

    doi: 10.3727/096504020X15929939001042

    MG-132 induces cell death in NSCLC cells. (A) H1299 and H441 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 1, and 5 μM) for 48 h. (B) A549 and H460 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 5, and 20 μM) for 48 h. Shown are representative images from three independent experiments. Scale bar: 400 μm. (C, D) Cell viability of H1299 and H441 cells were evaluated by cell counts. (E, F) Cell viability of A549 and H460 cells were evaluated by cell counts. Results are expressed as percent of cell viability normalized to control cells. The bar graphs represent mean with SD from three independent experiments. * p
    Figure Legend Snippet: MG-132 induces cell death in NSCLC cells. (A) H1299 and H441 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 1, and 5 μM) for 48 h. (B) A549 and H460 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 5, and 20 μM) for 48 h. Shown are representative images from three independent experiments. Scale bar: 400 μm. (C, D) Cell viability of H1299 and H441 cells were evaluated by cell counts. (E, F) Cell viability of A549 and H460 cells were evaluated by cell counts. Results are expressed as percent of cell viability normalized to control cells. The bar graphs represent mean with SD from three independent experiments. * p

    Techniques Used: Microscopy

    Proteasome inhibitors reduce c-Met expression in NSCLC cells. (A–D) H1299, H441, A549, and H460 cells were treated with increasing doses of bortezomib (0, 10, 50, and 100 nM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (E–H) H1299 and H441 cells were treated with increasing doses of MG-132 (0, 0.5, 1, 2, and 5 μM) for 48 h, while A549 and H460 cells were treated with higher doses of MG-132 (0, 5, 10, and 20 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (I–L) H1299, H441, A549, and H460 cells were treated with increasing doses of ONX-0914 (0, 1, 2, 5, and 10 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Shown are representative blots from three independent experiments. Intensities of blots were quantified by ImageJ software. Expression of c-Met was normalized to β-actin. The results are shown as mean ± SD of three independent experiments. * p
    Figure Legend Snippet: Proteasome inhibitors reduce c-Met expression in NSCLC cells. (A–D) H1299, H441, A549, and H460 cells were treated with increasing doses of bortezomib (0, 10, 50, and 100 nM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (E–H) H1299 and H441 cells were treated with increasing doses of MG-132 (0, 0.5, 1, 2, and 5 μM) for 48 h, while A549 and H460 cells were treated with higher doses of MG-132 (0, 5, 10, and 20 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (I–L) H1299, H441, A549, and H460 cells were treated with increasing doses of ONX-0914 (0, 1, 2, 5, and 10 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Shown are representative blots from three independent experiments. Intensities of blots were quantified by ImageJ software. Expression of c-Met was normalized to β-actin. The results are shown as mean ± SD of three independent experiments. * p

    Techniques Used: Expressing, Software

    Proteasome inhibitors increase PARP and caspase 3 cleavage and change p53 expression. (A) H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (B) H460 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (C) A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. Shown are representative blots from three independent experiments.
    Figure Legend Snippet: Proteasome inhibitors increase PARP and caspase 3 cleavage and change p53 expression. (A) H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (B) H460 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (C) A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. Shown are representative blots from three independent experiments.

    Techniques Used: Expressing

    The proteasome inhibitors have different effects on c-Met mRNA levels in NSCLC cells. (A–D) NSCLC cells were treated with 100 nM bortezomib for 48 h. c-Met mRNA levels were evaluated by real-time PCR. (E) c-Met mRNA levels in H1299 cells were evaluated after 5 μM MG-132 treatment for 48 h. (F) c-Met mRNA levels in H441 cells were evaluated after 2 μM MG-132 treatment for 48 h. (G) c-Met mRNA levels in H1299 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. (H) c-Met mRNA levels in H441 cells were evaluated after 5 μM ONX 0914 treatment for 48 h. (I) c-Met mRNA levels in H460 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. * p
    Figure Legend Snippet: The proteasome inhibitors have different effects on c-Met mRNA levels in NSCLC cells. (A–D) NSCLC cells were treated with 100 nM bortezomib for 48 h. c-Met mRNA levels were evaluated by real-time PCR. (E) c-Met mRNA levels in H1299 cells were evaluated after 5 μM MG-132 treatment for 48 h. (F) c-Met mRNA levels in H441 cells were evaluated after 2 μM MG-132 treatment for 48 h. (G) c-Met mRNA levels in H1299 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. (H) c-Met mRNA levels in H441 cells were evaluated after 5 μM ONX 0914 treatment for 48 h. (I) c-Met mRNA levels in H460 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. * p

    Techniques Used: Real-time Polymerase Chain Reaction

    Effect of proteasome inhibitors on CHX-mediated c-Met degradation. (A) H441 cells were treated with 20 μg/ml of CHX with or without MG132 (5 μM), bortezomib (100 nM), or ONX 0914 (10 μM). Cells were harvested at each time point over 0–8 h for immune blotting. Data represent one of three independent experiments with similar results. (B) Quantitative analysis of the immunoblots shown in (A) using ImageJ software. The results are shown as mean ± SD of three independent experiments. (C) A549 and H441 cells were transfected with c-Met-V5 plasmids for 1 day, and then were treated with proteasome inhibitors for an additional 48 h. H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM). A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM). Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Cell lysates were analyzed by immunoblotting with antibodies against V5 and β-actin.
    Figure Legend Snippet: Effect of proteasome inhibitors on CHX-mediated c-Met degradation. (A) H441 cells were treated with 20 μg/ml of CHX with or without MG132 (5 μM), bortezomib (100 nM), or ONX 0914 (10 μM). Cells were harvested at each time point over 0–8 h for immune blotting. Data represent one of three independent experiments with similar results. (B) Quantitative analysis of the immunoblots shown in (A) using ImageJ software. The results are shown as mean ± SD of three independent experiments. (C) A549 and H441 cells were transfected with c-Met-V5 plasmids for 1 day, and then were treated with proteasome inhibitors for an additional 48 h. H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM). A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM). Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Cell lysates were analyzed by immunoblotting with antibodies against V5 and β-actin.

    Techniques Used: Western Blot, Software, Transfection

    3) Product Images from "Production of Vesicular Stomatitis Virus Glycoprotein-Pseudotyped Lentiviral Vector Is Enhanced by Ezrin Silencing"

    Article Title: Production of Vesicular Stomatitis Virus Glycoprotein-Pseudotyped Lentiviral Vector Is Enhanced by Ezrin Silencing

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2020.00368

    HIV-1 Gag protein is digested in lysosomes and VSV-G rescues Gag protein from the digestion in COS7 cells. (A) COS7 cells were transfected with the HIV-1 Gag-Pol expression plasmid, and then were treated with DMSO, CMA, or MG-132. Cell lysates from the transfected cells were analyzed by western immunoblotting (upper panel). HIV-1 Gag precursor (p55) and mature capsid (p24) were indicated by arrows. Intensities of the p24 protein bands were measured by a densitometer. Relative values to the intensities in the pcDNA3.1-transfected cells are indicated (lower panel, n = 3). Asterisks show significant differences compared to DMSO. (B) COS7 cells were transfected with the HIV-1 Gag-Pol and VSV-G expression plasmids, and were treated with DMSO or CMA. Cell lysates prepared from the treated cells were analyzed by western blotting. Intensities of the p24 protein bands were measured by a densitometer. Relative values to the intensities in the pcDNA3.1-transfected cells are indicated (lower panel, n = 3). Asterisks show significant differences compared to DMSO. (C) AGM COS7 and human 293T cells were transfected with the MLV Gag-Pol expression plasmid together with pcDNA3.1 or VSV-G expression plasmid. Cell lysates prepared from the transfected cells were analyzed by western blotting. MLV Gag precursor (p65) and mature capsid (p30) were indicated by arrows.
    Figure Legend Snippet: HIV-1 Gag protein is digested in lysosomes and VSV-G rescues Gag protein from the digestion in COS7 cells. (A) COS7 cells were transfected with the HIV-1 Gag-Pol expression plasmid, and then were treated with DMSO, CMA, or MG-132. Cell lysates from the transfected cells were analyzed by western immunoblotting (upper panel). HIV-1 Gag precursor (p55) and mature capsid (p24) were indicated by arrows. Intensities of the p24 protein bands were measured by a densitometer. Relative values to the intensities in the pcDNA3.1-transfected cells are indicated (lower panel, n = 3). Asterisks show significant differences compared to DMSO. (B) COS7 cells were transfected with the HIV-1 Gag-Pol and VSV-G expression plasmids, and were treated with DMSO or CMA. Cell lysates prepared from the treated cells were analyzed by western blotting. Intensities of the p24 protein bands were measured by a densitometer. Relative values to the intensities in the pcDNA3.1-transfected cells are indicated (lower panel, n = 3). Asterisks show significant differences compared to DMSO. (C) AGM COS7 and human 293T cells were transfected with the MLV Gag-Pol expression plasmid together with pcDNA3.1 or VSV-G expression plasmid. Cell lysates prepared from the transfected cells were analyzed by western blotting. MLV Gag precursor (p65) and mature capsid (p30) were indicated by arrows.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Western Blot

    4) Product Images from "Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct"

    Article Title: Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct

    Journal: Cells

    doi: 10.3390/cells9051208

    Semiquantitative immunoblotting of SNX27 ( A , B ; F , G ; and K , L ) and AQP2 ( A , C ; F , H ; and K , M ) in total cell lysates from mpkCCDc14 cells transfected with control-siRNA or SNX27-siRNA. Semiquantitative immunoblotting of AQP2 in total cell lysates from mpkCCDc14 cells transfected with control siRNA or SNX27-siRNA subjected to 24-h dDAVP stimulation (10 −9 M) or 3-h withdrawal (Withdrawal) after dDAVP stimulation (10 −9 M, 24 h) in the absence (-) or the presence (+) of chloroquine (10 −4 M, for the last 3 h, ( A , D , E )), bafilomycin (10 −7 M, for the last 3 h, ( F , I , J )), or MG-132 treatment (MG-132, 10 −6 M, for the last 3 h, ( K , N , O )). n indicates the number of cell preparations from three independent experiments. D, dDAVP. * p
    Figure Legend Snippet: Semiquantitative immunoblotting of SNX27 ( A , B ; F , G ; and K , L ) and AQP2 ( A , C ; F , H ; and K , M ) in total cell lysates from mpkCCDc14 cells transfected with control-siRNA or SNX27-siRNA. Semiquantitative immunoblotting of AQP2 in total cell lysates from mpkCCDc14 cells transfected with control siRNA or SNX27-siRNA subjected to 24-h dDAVP stimulation (10 −9 M) or 3-h withdrawal (Withdrawal) after dDAVP stimulation (10 −9 M, 24 h) in the absence (-) or the presence (+) of chloroquine (10 −4 M, for the last 3 h, ( A , D , E )), bafilomycin (10 −7 M, for the last 3 h, ( F , I , J )), or MG-132 treatment (MG-132, 10 −6 M, for the last 3 h, ( K , N , O )). n indicates the number of cell preparations from three independent experiments. D, dDAVP. * p

    Techniques Used: Transfection

    5) Product Images from "The intrinsic chaperone network of Arabidopsis stem cells confers protection against proteotoxic stress). The intrinsic chaperone network of Arabidopsis stem cells confers protection against proteotoxic stress"

    Article Title: The intrinsic chaperone network of Arabidopsis stem cells confers protection against proteotoxic stress). The intrinsic chaperone network of Arabidopsis stem cells confers protection against proteotoxic stress

    Journal: Aging Cell

    doi: 10.1111/acel.13446

    Proteotoxic stress causes differential protein aggregation in the distinct cell types of the root. (a) Representative images of wild‐type plant roots grown under control (22°C or 22°C + DMSO) or proteotoxic stress conditions stained with ProteoStat Aggresome Detection kit. ProteoStat (red, protein aggregates), Hoechst (blue, nuclei), and Merge (ProteoStat and Hoechst) images are shown. For heat stress assay, 4 days after germination (DAG) plants were transferred to 37°C for 2 days. For proteasome inhibition experiments, plants were germinated and grown in plates supplemented with 30 µM MG‐132 and analyzed at 6 DAG stage. White bars represent 100 µm. (b) Relative ProteoStat fluorescence levels comparing the area containing proximal (close to the QC) and distal cells from roots under proteotoxic stress (areas used for quantification are indicated with white dotted ovals in Figure 1a ). Scatter plots represent mean ± s.e.m of four independent experiments. The statistical comparisons were made by two‐tailed Student's t test for unpaired samples. p values: * p
    Figure Legend Snippet: Proteotoxic stress causes differential protein aggregation in the distinct cell types of the root. (a) Representative images of wild‐type plant roots grown under control (22°C or 22°C + DMSO) or proteotoxic stress conditions stained with ProteoStat Aggresome Detection kit. ProteoStat (red, protein aggregates), Hoechst (blue, nuclei), and Merge (ProteoStat and Hoechst) images are shown. For heat stress assay, 4 days after germination (DAG) plants were transferred to 37°C for 2 days. For proteasome inhibition experiments, plants were germinated and grown in plates supplemented with 30 µM MG‐132 and analyzed at 6 DAG stage. White bars represent 100 µm. (b) Relative ProteoStat fluorescence levels comparing the area containing proximal (close to the QC) and distal cells from roots under proteotoxic stress (areas used for quantification are indicated with white dotted ovals in Figure 1a ). Scatter plots represent mean ± s.e.m of four independent experiments. The statistical comparisons were made by two‐tailed Student's t test for unpaired samples. p values: * p

    Techniques Used: Staining, Inhibition, Fluorescence, Two Tailed Test

    Overexpression of CCT8 is sufficient to ameliorate protein aggregation in differentiated cells and confer resistance to proteotoxic stress in plants (a) Representative images of ProteoStat staining of 6 DAG Col‐0 and 35S : CCT8 germinated and grown in plates supplemented with 15 µM MG‐132. Scale bar represents 100 µm. (b) Relative ProteoStat fluorescence levels comparing the areas depicted with white dotted circles in Figure 4a . Graph represents mean ± s.e.m of four independent experiments. (c) Root lengths of MG‐132‐treated plants relative to their corresponding controls treated with DMSO. Statistical comparisons in (b–c) were made by two‐tailed Student's t test for unpaired samples. p value: ** p
    Figure Legend Snippet: Overexpression of CCT8 is sufficient to ameliorate protein aggregation in differentiated cells and confer resistance to proteotoxic stress in plants (a) Representative images of ProteoStat staining of 6 DAG Col‐0 and 35S : CCT8 germinated and grown in plates supplemented with 15 µM MG‐132. Scale bar represents 100 µm. (b) Relative ProteoStat fluorescence levels comparing the areas depicted with white dotted circles in Figure 4a . Graph represents mean ± s.e.m of four independent experiments. (c) Root lengths of MG‐132‐treated plants relative to their corresponding controls treated with DMSO. Statistical comparisons in (b–c) were made by two‐tailed Student's t test for unpaired samples. p value: ** p

    Techniques Used: Over Expression, Staining, Fluorescence, Two Tailed Test

    6) Product Images from "Role of Ubiquitin and Proteasomes in Phagosome Maturation"

    Article Title: Role of Ubiquitin and Proteasomes in Phagosome Maturation

    Journal:

    doi: 10.1091/mbc.E04-06-0464

    Proteasome inhibition prevents loss of FcγRIIA from the phagosome. CHO cells stably transfected with FcγRIIA-GFP were either left untreated (A) or pretreated with 10 μM MG-132 for 3 h (C) before phagocytosis of opsonized beads
    Figure Legend Snippet: Proteasome inhibition prevents loss of FcγRIIA from the phagosome. CHO cells stably transfected with FcγRIIA-GFP were either left untreated (A) or pretreated with 10 μM MG-132 for 3 h (C) before phagocytosis of opsonized beads

    Techniques Used: Inhibition, Stable Transfection, Transfection

    Proteasome inhibition has no effect on fusion of phagosomes with MVB or lysosomes. RAW macrophages were either left untreated (A, D, and G) or were pretreated with 10 μM MG-132 for 1.5 h (B, E, and H) and allowed to ingest opsonized beads for
    Figure Legend Snippet: Proteasome inhibition has no effect on fusion of phagosomes with MVB or lysosomes. RAW macrophages were either left untreated (A, D, and G) or were pretreated with 10 μM MG-132 for 1.5 h (B, E, and H) and allowed to ingest opsonized beads for

    Techniques Used: Inhibition

    The effects of proteasome inhibition on fission are not through depletion of cellular free ubiquitin. (A) RAW macrophages were either left untreated (control) or were pretreated with MG-132 for 1.5 h and then allowed to ingest opsonized beads for 12 min
    Figure Legend Snippet: The effects of proteasome inhibition on fission are not through depletion of cellular free ubiquitin. (A) RAW macrophages were either left untreated (control) or were pretreated with MG-132 for 1.5 h and then allowed to ingest opsonized beads for 12 min

    Techniques Used: Inhibition

    Translocation of the cytosolic tail of FcγRIIA to an acidic compartment is prevented by proteasome inhibitors. CHO cells stably transfected with FcγRIIA-GFP were pretreated with 10 μM MG-132 for 3 h and allowed to ingest opsonized
    Figure Legend Snippet: Translocation of the cytosolic tail of FcγRIIA to an acidic compartment is prevented by proteasome inhibitors. CHO cells stably transfected with FcγRIIA-GFP were pretreated with 10 μM MG-132 for 3 h and allowed to ingest opsonized

    Techniques Used: Translocation Assay, Stable Transfection, Transfection

    7) Product Images from "The human-specific paralogs SRGAP2B and SRGAP2C differentially modulate SRGAP2A-dependent synaptic development"

    Article Title: The human-specific paralogs SRGAP2B and SRGAP2C differentially modulate SRGAP2A-dependent synaptic development

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-54887-4

    Proteasome degradation of SRGAP2 proteins. ( A ) Expression of RFP-tagged F-BARx, F-BAR Δ49 , or SRGAP2C in mouse cortical pyramidal neurons cultured for 18–21 days in vitro (DIV). Low levels of expression were observed for F-BAR Δ49 and SRGAP2C, while addition of the proteasome inhibitor MG-132 resulted in a strong increase after 8 h of treatment. F-BARx is highly expressed throughout the experiment. Scale bar top panels: 25 µm, bottom panels: 5 µm. ( B ) Quantification of fluorescence intensity after MG-132 treatment. n F-BARx = 31 neurons, n F-BAR Δ49 = 50 neurons, n SRGAP2C = 47 neurons; multiple t tests with Holm-Sidak correction for multiple comparisons (α = 0.05); ****p
    Figure Legend Snippet: Proteasome degradation of SRGAP2 proteins. ( A ) Expression of RFP-tagged F-BARx, F-BAR Δ49 , or SRGAP2C in mouse cortical pyramidal neurons cultured for 18–21 days in vitro (DIV). Low levels of expression were observed for F-BAR Δ49 and SRGAP2C, while addition of the proteasome inhibitor MG-132 resulted in a strong increase after 8 h of treatment. F-BARx is highly expressed throughout the experiment. Scale bar top panels: 25 µm, bottom panels: 5 µm. ( B ) Quantification of fluorescence intensity after MG-132 treatment. n F-BARx = 31 neurons, n F-BAR Δ49 = 50 neurons, n SRGAP2C = 47 neurons; multiple t tests with Holm-Sidak correction for multiple comparisons (α = 0.05); ****p

    Techniques Used: Expressing, Cell Culture, In Vitro, Fluorescence

    8) Product Images from "Modulation of inflammatory response in sepsis by proteasome inhibition"

    Article Title: Modulation of inflammatory response in sepsis by proteasome inhibition

    Journal:

    doi: 10.1111/j.1365-2613.2006.00490.x

    Survival curves. Groups: caecal ligation and puncture (CLP) only, MG-132 administration 3 h before CLP, and MG-132 administration 3 h after CLP. In MG-132 treated groups, survival was significantly longer compared with CLP group. Logrank test, P = 0.011,
    Figure Legend Snippet: Survival curves. Groups: caecal ligation and puncture (CLP) only, MG-132 administration 3 h before CLP, and MG-132 administration 3 h after CLP. In MG-132 treated groups, survival was significantly longer compared with CLP group. Logrank test, P = 0.011,

    Techniques Used: Ligation

    9) Product Images from "Absence of Mal/TIRAP Results in Abrogated Imidazoquinolinones-Dependent Activation of IRF7 and Suppressed IFNβ and IFN-I Activated Gene Production"

    Article Title: Absence of Mal/TIRAP Results in Abrogated Imidazoquinolinones-Dependent Activation of IRF7 and Suppressed IFNβ and IFN-I Activated Gene Production

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21238925

    Selective agonist of TLR7 induces expression of IFNβ and IP-10 in human pmDC05 cell line in Mal dependent manner. Human pmDC05 cells were pretreated with control or Mal blocking peptide (20 µM) for 16h. Next, cells were pretreated with DMSO (control), FR180204 (2 µM) ( C , D ) or JSH-23 (10 µM) and MG-132 (5 µM) ( E , F ) for 30 min. Thereafter, cells were treated with R837 (10 µg/mL) for 4 h. Total RNA was isolated, converted to first-strand cDNA, and used as a template for quantitative real-time RT-PCR as described under “Materials and Methods.” Quantitative real-time RT-PCR was used to assay the expression levels of IFNβ ( A , C , E ), IP-10 ( B , D , F ). Experiments were repeated at least three times and data are presented in relative expression units where HPRT was used to normalize all samples and DMSO treated cells (control) were assigned an arbitrary value of 1. * p ≤ 0.001; *** p ≤ 0.05.
    Figure Legend Snippet: Selective agonist of TLR7 induces expression of IFNβ and IP-10 in human pmDC05 cell line in Mal dependent manner. Human pmDC05 cells were pretreated with control or Mal blocking peptide (20 µM) for 16h. Next, cells were pretreated with DMSO (control), FR180204 (2 µM) ( C , D ) or JSH-23 (10 µM) and MG-132 (5 µM) ( E , F ) for 30 min. Thereafter, cells were treated with R837 (10 µg/mL) for 4 h. Total RNA was isolated, converted to first-strand cDNA, and used as a template for quantitative real-time RT-PCR as described under “Materials and Methods.” Quantitative real-time RT-PCR was used to assay the expression levels of IFNβ ( A , C , E ), IP-10 ( B , D , F ). Experiments were repeated at least three times and data are presented in relative expression units where HPRT was used to normalize all samples and DMSO treated cells (control) were assigned an arbitrary value of 1. * p ≤ 0.001; *** p ≤ 0.05.

    Techniques Used: Expressing, Blocking Assay, Isolation, Quantitative RT-PCR

    10) Product Images from "2,3,7,8-Tetrachlorodibenzo-p-dioxin poly(ADP-ribose) polymerase (TiPARP, ARTD14) is a mono-ADP-ribosyltransferase and repressor of aryl hydrocarbon receptor transactivation"

    Article Title: 2,3,7,8-Tetrachlorodibenzo-p-dioxin poly(ADP-ribose) polymerase (TiPARP, ARTD14) is a mono-ADP-ribosyltransferase and repressor of aryl hydrocarbon receptor transactivation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1337

    TiPARP is a labile negative regulator of AHR. ( A ) TiPARP mRNA was rapidly degraded after actinomycin D treatment. T-47D cells were treated with 1 µg/ml actinomycin D at the times indicated and Tiparp mRNA expression levels determined by qPCR. ( B ) GFP-TiPARP overexpression was increased after proteasome inhibition. HuH-7 cells were transfected with pEGFP-TiPARP, pcDNA (vector) and pEGFP for 24 h and treated with 25 µM MG-132 for 6 h, and GFP-TiPARP protein levels were determined by western blot using anti-GFP antibody. The data were from a representative western blot from three independent experiments. ( C ) Ahr target gene induction in MEF lines pre-treated with 10 µg/ml CHX. MEF cell lines were pre-treated with 10 µg/ml CHX for 1 h, then treated with TCDD for 6 h and gene expression was determined. Data were normalized to wildtype DMSO. Fold changes between TCDD alone and CHX + TCDD (CHX + T) were provided for each gene. Gene expression results were shown as means ± S.E.M for three independent experiments and significance analysed by one-way ANOVA and Tukey’s multiple co mparisons test. Gene expression levels significantly different ( P
    Figure Legend Snippet: TiPARP is a labile negative regulator of AHR. ( A ) TiPARP mRNA was rapidly degraded after actinomycin D treatment. T-47D cells were treated with 1 µg/ml actinomycin D at the times indicated and Tiparp mRNA expression levels determined by qPCR. ( B ) GFP-TiPARP overexpression was increased after proteasome inhibition. HuH-7 cells were transfected with pEGFP-TiPARP, pcDNA (vector) and pEGFP for 24 h and treated with 25 µM MG-132 for 6 h, and GFP-TiPARP protein levels were determined by western blot using anti-GFP antibody. The data were from a representative western blot from three independent experiments. ( C ) Ahr target gene induction in MEF lines pre-treated with 10 µg/ml CHX. MEF cell lines were pre-treated with 10 µg/ml CHX for 1 h, then treated with TCDD for 6 h and gene expression was determined. Data were normalized to wildtype DMSO. Fold changes between TCDD alone and CHX + TCDD (CHX + T) were provided for each gene. Gene expression results were shown as means ± S.E.M for three independent experiments and significance analysed by one-way ANOVA and Tukey’s multiple co mparisons test. Gene expression levels significantly different ( P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Over Expression, Inhibition, Transfection, Plasmid Preparation, Western Blot

    11) Product Images from "Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis"

    Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

    Journal: bioRxiv

    doi: 10.1101/2021.08.12.456108

    Deubiquitinase USP7 prevents ubiquitination-mediated degradation of XIAP in p53 independent fashion. HEK cells were used in experiments A-E, H L and mentioned in other cases. A, Cells co-transfected with HA-Ub and GFP-XIAP and 24 hrs post-transfection cells were further treated with MG-132 (25µM) and vehicle control for an additional 4 hrs. GFP-tagged proteins were pulled down from lysates using GFP antibody and Protein-A Sepharose bead; analyzed by IB using indicated antibodies. B, Cells were transfected with indicated constructs; 24 hrs post-transfection, cells were treated with P5091 (15µM) for another 24 hrs. Before harvesting, the cells were further treated with MG-132 for 4 hrs. Lysates were prepared and used for pull-down using GFP antibody followed by IB using indicated antibodies. C, Cells were transfected individually with EV, USP7, and USP7 C223S plasmids. Pull-down assay was performed using XIAP antibody, bead-bound proteins and total cell lysate were analyzed by IB to detect the change in poly-ubiquitination pattern. D, Cells were transfected with indicated plasmids; 24 hr post-transfection, treated with P5091 (15µM) and vehicle control for another 24 hours. Cells were harvested after MG-132 treatment for further 4 hrs, followed by pull-down assay was performed using GFP antibody and IB with indicated antibodies. E, Cells were transfected with either WT or different Ub mutants as depicted in the figure. Following lysate preparation, pulled down the proteins using GFP antibody followed by IB using the indicated antibodies. Input (3%) was run separately for Control. F, Cell lysates prepared from p53 WT and p53 null HCT116 cells subjected to IB with the indicated antibodies. G, HCT116 (p53 wt ) cells were treated with Nutlin3A (5µM) for 24 hrs, the protein level of XIAP and other p53 responsive genes were analyzed by IB. H, Cells were treated with Nutlin3A in a dose-dependent manner (5µM and 10µM) to analyze the level of indicated proteins by IB. I, HCT116 (p53 wt ) and HT29 (p53 mut ) cells were treated with P5091 in a dose-dependent manner (0, 10, and 20µM) for 24 hrs and check the levels of the indicated proteins by IB. J, Expression of indicated genes in HCT116 cells containing either p53 wt or p53 mut was analyzed by qRT-PCR. K, HCT116 cells were treated with Doxorubicin (10µM) for 3 hrs, washed and kept in fresh media without Dox for another 21hrs before harvesting them. Expression (mRNA level) of the indicated genes was analyzed by qRT-PCR. L, Cells were transfected with either EV (left panel) or GFP-USP7 (right panel) for 24 hrs followed by Dox treatment in a dose-dependent manner (0, 1, 2, 3µM) for another 24 hrs. Expression of the indicated proteins was analyzed by IB. Expression of XIAP was normalized against GAPDH and plotted here. The data are representative of three biological replicates. qRT-PCR data represents the mean ± SD of three independent biological replicates. Indicated P-values were calculated using Student’s t-test and P
    Figure Legend Snippet: Deubiquitinase USP7 prevents ubiquitination-mediated degradation of XIAP in p53 independent fashion. HEK cells were used in experiments A-E, H L and mentioned in other cases. A, Cells co-transfected with HA-Ub and GFP-XIAP and 24 hrs post-transfection cells were further treated with MG-132 (25µM) and vehicle control for an additional 4 hrs. GFP-tagged proteins were pulled down from lysates using GFP antibody and Protein-A Sepharose bead; analyzed by IB using indicated antibodies. B, Cells were transfected with indicated constructs; 24 hrs post-transfection, cells were treated with P5091 (15µM) for another 24 hrs. Before harvesting, the cells were further treated with MG-132 for 4 hrs. Lysates were prepared and used for pull-down using GFP antibody followed by IB using indicated antibodies. C, Cells were transfected individually with EV, USP7, and USP7 C223S plasmids. Pull-down assay was performed using XIAP antibody, bead-bound proteins and total cell lysate were analyzed by IB to detect the change in poly-ubiquitination pattern. D, Cells were transfected with indicated plasmids; 24 hr post-transfection, treated with P5091 (15µM) and vehicle control for another 24 hours. Cells were harvested after MG-132 treatment for further 4 hrs, followed by pull-down assay was performed using GFP antibody and IB with indicated antibodies. E, Cells were transfected with either WT or different Ub mutants as depicted in the figure. Following lysate preparation, pulled down the proteins using GFP antibody followed by IB using the indicated antibodies. Input (3%) was run separately for Control. F, Cell lysates prepared from p53 WT and p53 null HCT116 cells subjected to IB with the indicated antibodies. G, HCT116 (p53 wt ) cells were treated with Nutlin3A (5µM) for 24 hrs, the protein level of XIAP and other p53 responsive genes were analyzed by IB. H, Cells were treated with Nutlin3A in a dose-dependent manner (5µM and 10µM) to analyze the level of indicated proteins by IB. I, HCT116 (p53 wt ) and HT29 (p53 mut ) cells were treated with P5091 in a dose-dependent manner (0, 10, and 20µM) for 24 hrs and check the levels of the indicated proteins by IB. J, Expression of indicated genes in HCT116 cells containing either p53 wt or p53 mut was analyzed by qRT-PCR. K, HCT116 cells were treated with Doxorubicin (10µM) for 3 hrs, washed and kept in fresh media without Dox for another 21hrs before harvesting them. Expression (mRNA level) of the indicated genes was analyzed by qRT-PCR. L, Cells were transfected with either EV (left panel) or GFP-USP7 (right panel) for 24 hrs followed by Dox treatment in a dose-dependent manner (0, 1, 2, 3µM) for another 24 hrs. Expression of the indicated proteins was analyzed by IB. Expression of XIAP was normalized against GAPDH and plotted here. The data are representative of three biological replicates. qRT-PCR data represents the mean ± SD of three independent biological replicates. Indicated P-values were calculated using Student’s t-test and P

    Techniques Used: Transfection, Construct, Pull Down Assay, Expressing, Quantitative RT-PCR

    12) Product Images from "Cyclin C promoter occupancy directs changes in stress-dependent transcription"

    Article Title: Cyclin C promoter occupancy directs changes in stress-dependent transcription

    Journal: bioRxiv

    doi: 10.1101/2020.07.14.202580

    Release of cyclin C from promoters does not require Medl3/Medl3L degradation. RT-qPCR (A) and ChIP analysis (B) for the cyclin C repressed genes indicated following H 2 O 2 /MG-132 treatment. ChIP data are shown as percent input DNA compared to a non-specific control (GFP antibody). H 2 O 2 treated WT control is from Fig. 1 to aid in comparing results. (C) Med13 and Med13L levels were monitored by Western blot analysis in MEFs following exposure to H 2 O 2 and MG-132 as indicated, β-actin was used as the internal loading control. (D) The ratios of Med13 and Med13L signals to the β-actin internal control were calculated then compared WT untreated control (n=3). Statistical significance is indicated by the following: * p-value
    Figure Legend Snippet: Release of cyclin C from promoters does not require Medl3/Medl3L degradation. RT-qPCR (A) and ChIP analysis (B) for the cyclin C repressed genes indicated following H 2 O 2 /MG-132 treatment. ChIP data are shown as percent input DNA compared to a non-specific control (GFP antibody). H 2 O 2 treated WT control is from Fig. 1 to aid in comparing results. (C) Med13 and Med13L levels were monitored by Western blot analysis in MEFs following exposure to H 2 O 2 and MG-132 as indicated, β-actin was used as the internal loading control. (D) The ratios of Med13 and Med13L signals to the β-actin internal control were calculated then compared WT untreated control (n=3). Statistical significance is indicated by the following: * p-value

    Techniques Used: Quantitative RT-PCR, Chromatin Immunoprecipitation, Western Blot

    13) Product Images from "Ras transformation results in cleavage of reticulon protein Nogo-B that is associated with impairment of IFN response"

    Article Title: Ras transformation results in cleavage of reticulon protein Nogo-B that is associated with impairment of IFN response

    Journal: Cell Cycle

    doi: 10.1080/15384101.2015.1044187

    N-terminal of Nogo-(B)is cleaved in Ras-transformed cells. ( A ) Comparison of cell surface proteins between non-transformed and Ras-transformed NIH-3T3 cells. Cell surface proteins were labeled with membrane impermeable biotin, then isolated using streptavidin-conjugated magnetic beads. Isolated proteins were analyzed by SDS-PAGE/silver nitrate staining. Bands showing different intensity levels were excised and identified by mass spectrometry. Arrows indicate proteins containing Nogo protein peptides. The 43 kDa band(s) represents Nogo-B, and the lower band (˜34 kDa) likely represents its cleavage product. ( B ) Schematic diagram of Nogo isoforms. Three major isoforms of Nogo (A, B, and C) share C-terminal region (shaded in black). Nogo-A and -B share N-terminal region of 167 a.a. (light gray). Peptide sequences detected by mass spectrometry were shown in Nogo-B structure. Antibodies used for detecting C-terminal and N-terminal ends of Nogo were indicated ('Y' in black and for C-terminus and 'Y' in light gray for N-terminus). ( C ) Detection of Nogo-B in total cell lysates (left panel) and membrane fractions (right panel) from non- and Ras-transformed cells by western blot using the antibody detecting the C-terminus of Nogo-B. β-actin and Calnexin (ER membrane marker) were used as loading controls. ( D ) Cell lysate of non- or Ras-transformed cells were fractionated by differential centrifugation (Lys, lysate; Nuc, nucleus; Mit, mitochondrial fraction; Cyt, cytosolic fraction; Mic, microsomal fraction). Western blots were carried out using the anti-C-terminal antibody (upper panel), or the anti-N-terminal antibody (lower panel). Cox IV, Calnexin, and β-actin were used as mitochondrial, ER, and cytosol markers, respectively. ( E ) mRNA levels of Nogo-B in non-transformed and Ras-transformed cells were compared using RT-PCR with Nogo-B specific primers. GAPDH was used as a loading control. ( F ) Ras-transformed cells were treated with vehicle control (DMSO), caspase inhibitor (Z-VAD-FMK, 10 μM), proteasome inhibitor (MG-132, 1 μM) or both for 18 hrs. Full-length Nogo-B is indicated as “FL” and a cleavage product as “C.”
    Figure Legend Snippet: N-terminal of Nogo-(B)is cleaved in Ras-transformed cells. ( A ) Comparison of cell surface proteins between non-transformed and Ras-transformed NIH-3T3 cells. Cell surface proteins were labeled with membrane impermeable biotin, then isolated using streptavidin-conjugated magnetic beads. Isolated proteins were analyzed by SDS-PAGE/silver nitrate staining. Bands showing different intensity levels were excised and identified by mass spectrometry. Arrows indicate proteins containing Nogo protein peptides. The 43 kDa band(s) represents Nogo-B, and the lower band (˜34 kDa) likely represents its cleavage product. ( B ) Schematic diagram of Nogo isoforms. Three major isoforms of Nogo (A, B, and C) share C-terminal region (shaded in black). Nogo-A and -B share N-terminal region of 167 a.a. (light gray). Peptide sequences detected by mass spectrometry were shown in Nogo-B structure. Antibodies used for detecting C-terminal and N-terminal ends of Nogo were indicated ('Y' in black and for C-terminus and 'Y' in light gray for N-terminus). ( C ) Detection of Nogo-B in total cell lysates (left panel) and membrane fractions (right panel) from non- and Ras-transformed cells by western blot using the antibody detecting the C-terminus of Nogo-B. β-actin and Calnexin (ER membrane marker) were used as loading controls. ( D ) Cell lysate of non- or Ras-transformed cells were fractionated by differential centrifugation (Lys, lysate; Nuc, nucleus; Mit, mitochondrial fraction; Cyt, cytosolic fraction; Mic, microsomal fraction). Western blots were carried out using the anti-C-terminal antibody (upper panel), or the anti-N-terminal antibody (lower panel). Cox IV, Calnexin, and β-actin were used as mitochondrial, ER, and cytosol markers, respectively. ( E ) mRNA levels of Nogo-B in non-transformed and Ras-transformed cells were compared using RT-PCR with Nogo-B specific primers. GAPDH was used as a loading control. ( F ) Ras-transformed cells were treated with vehicle control (DMSO), caspase inhibitor (Z-VAD-FMK, 10 μM), proteasome inhibitor (MG-132, 1 μM) or both for 18 hrs. Full-length Nogo-B is indicated as “FL” and a cleavage product as “C.”

    Techniques Used: Polyacrylamide Gel Electrophoresis, Transformation Assay, Labeling, Isolation, Magnetic Beads, SDS Page, Staining, Mass Spectrometry, Western Blot, Marker, Centrifugation, Reverse Transcription Polymerase Chain Reaction

    14) Product Images from "COP9 Signalosome- and 26S Proteasome-dependent Regulation of SCFTIR1 Accumulation inArabidopsis * * S⃞"

    Article Title: COP9 Signalosome- and 26S Proteasome-dependent Regulation of SCFTIR1 Accumulation inArabidopsis * * S⃞

    Journal:

    doi: 10.1074/jbc.M809069200

    Post-translational regulation of SCF TIR1 accumulation in wild-type plants. L er np :: TIR1-HAStrep seedlings were grown in liquid MS for 7 days. A , seedlings were incubated for 6 h with the proteasome inhibitor MG-132 (50 μ m (+) or with DMSO
    Figure Legend Snippet: Post-translational regulation of SCF TIR1 accumulation in wild-type plants. L er np :: TIR1-HAStrep seedlings were grown in liquid MS for 7 days. A , seedlings were incubated for 6 h with the proteasome inhibitor MG-132 (50 μ m (+) or with DMSO

    Techniques Used: Incubation

    15) Product Images from "Hypoxia differentially regulates estrogen receptor alpha in 2D and 3D culture formats"

    Article Title: Hypoxia differentially regulates estrogen receptor alpha in 2D and 3D culture formats

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2019.05.025

    2D and 3D T47D cultures exposed to normoxic or hypoxic conditions in E2-deprived (Veh) or -supplemented (E2) medium for 24 h. Total RNA was extracted and relative expression of ESR1 (A, B) was determined using the ΔΔC t method; β-actin served as the reference gene. Data represent the average ± SEM, from n = 9 replicate cultures from three different cell passages. 2D and 3D T47D cultures were exposed to normoxic or hypoxic conditions in estrogen-deprived medium in the presence or absence of MG-132 (10 μM) for 8 h. HIF-1α (C, D) and ERα (E, F) protein levels were quantified with ELISA. Data represent the average ± SEM, from n ≥ 6 replicate cultures from two different cell passages. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.
    Figure Legend Snippet: 2D and 3D T47D cultures exposed to normoxic or hypoxic conditions in E2-deprived (Veh) or -supplemented (E2) medium for 24 h. Total RNA was extracted and relative expression of ESR1 (A, B) was determined using the ΔΔC t method; β-actin served as the reference gene. Data represent the average ± SEM, from n = 9 replicate cultures from three different cell passages. 2D and 3D T47D cultures were exposed to normoxic or hypoxic conditions in estrogen-deprived medium in the presence or absence of MG-132 (10 μM) for 8 h. HIF-1α (C, D) and ERα (E, F) protein levels were quantified with ELISA. Data represent the average ± SEM, from n ≥ 6 replicate cultures from two different cell passages. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    16) Product Images from "LSD1-mediated demethylation of OCT4 safeguards pluripotent stem cells by maintaining the transcription of PORE-motif-containing genes"

    Article Title: LSD1-mediated demethylation of OCT4 safeguards pluripotent stem cells by maintaining the transcription of PORE-motif-containing genes

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-89734-y

    K222 methylation promotes proteasome-independent degradation of OCT4. ( A ) NCCIT cells were infected with LSD1 shRNA lentiviruses. After 96 h, cells were pre-treated with 5 μM MG-132 or vehicle for 1 h, followed by addition of 20 μg/ml CHX and further incubation for 0 to 12 h in the presence of MG-132. Whole cell lysates were immunoblotted with the indicated antibodies. The curves represented quantitation of the OCT4 protein levels. ( B ) NCCIT cells were pre-treated with 100 μM 2-PCPA and 5 μM MG-132 for 1 h and followed by the same steps as described in ( A ). ( C ) NCCIT cells pre-treated with DMSO (left panels) or 2-PCPA (right panels) were exposed to CHX for 0 to 24 h, the whole cell lysates were immunoblotted with the indicated antibodies, and the OCT4 protein levels were quantified in ( D ). ( E ) U87 cells were transfected with the shOCT4-FLAG-OCT4 plasmids (WT and variants). After 72 h, they were treated with 20 μg/ml CHX for 0 to 24 h, the whole cell lysates were immunoblotted with the indicated antibodies, and the OCT4 protein levels were quantified in ( F ). ( G ) NCCIT cells were pre-treated with 5 μM MG-132 or 200 μM chloroquine singly or in combination for 1 h followed by addition of 20 μg/ml CHX and further incubation for 0–24 h. Whole cell lysates were immunoblotted with the indicated antibodies. (H) NCCIT cells were pre-treated with 100 μM 2-PCPA alone or in combination with 200 μM chloroquine for 1 h followed by addition of 20 μg/ml CHX and further incubation for 0 to 24 h. Whole cell lysates were immunoblotted with the indicated antibodies, and the OCT4 protein levels were quantified in ( I ). Results shown in ( D ) and ( F ) were presented as means ± S.D. of triplicate measurements from single experiment representative of 3 independent experiments. Two-tailed unpaired Student’s t tests were used for statistical analyses. *P
    Figure Legend Snippet: K222 methylation promotes proteasome-independent degradation of OCT4. ( A ) NCCIT cells were infected with LSD1 shRNA lentiviruses. After 96 h, cells were pre-treated with 5 μM MG-132 or vehicle for 1 h, followed by addition of 20 μg/ml CHX and further incubation for 0 to 12 h in the presence of MG-132. Whole cell lysates were immunoblotted with the indicated antibodies. The curves represented quantitation of the OCT4 protein levels. ( B ) NCCIT cells were pre-treated with 100 μM 2-PCPA and 5 μM MG-132 for 1 h and followed by the same steps as described in ( A ). ( C ) NCCIT cells pre-treated with DMSO (left panels) or 2-PCPA (right panels) were exposed to CHX for 0 to 24 h, the whole cell lysates were immunoblotted with the indicated antibodies, and the OCT4 protein levels were quantified in ( D ). ( E ) U87 cells were transfected with the shOCT4-FLAG-OCT4 plasmids (WT and variants). After 72 h, they were treated with 20 μg/ml CHX for 0 to 24 h, the whole cell lysates were immunoblotted with the indicated antibodies, and the OCT4 protein levels were quantified in ( F ). ( G ) NCCIT cells were pre-treated with 5 μM MG-132 or 200 μM chloroquine singly or in combination for 1 h followed by addition of 20 μg/ml CHX and further incubation for 0–24 h. Whole cell lysates were immunoblotted with the indicated antibodies. (H) NCCIT cells were pre-treated with 100 μM 2-PCPA alone or in combination with 200 μM chloroquine for 1 h followed by addition of 20 μg/ml CHX and further incubation for 0 to 24 h. Whole cell lysates were immunoblotted with the indicated antibodies, and the OCT4 protein levels were quantified in ( I ). Results shown in ( D ) and ( F ) were presented as means ± S.D. of triplicate measurements from single experiment representative of 3 independent experiments. Two-tailed unpaired Student’s t tests were used for statistical analyses. *P

    Techniques Used: Methylation, Infection, shRNA, Incubation, Quantitation Assay, Transfection, Two Tailed Test

    17) Product Images from "Non-canonical autophagy in dendritic cells restricts cross-presentation and anti-tumor immunity"

    Article Title: Non-canonical autophagy in dendritic cells restricts cross-presentation and anti-tumor immunity

    Journal: bioRxiv

    doi: 10.1101/789867

    Rubcn -/- DCs exhibit increased phagosome-to-cytosol escape and proteasome-mediated generation of peptides. Bone marrow-derived dendritic cells (DCs) were generated from Rubcn +/+ (black) and Rubcn -/- (red) mice in vitro with FLT3-L for 7 days. ( A-B ) DCs were loaded with 1 µm CCF4, then co-cultured with apoptotic B16-OVA in the absence or presence of β-lactamase (2 mg/ml) for 90 or 180 minutes at 4°C or 37°C. Uncleaved CCF4 was measured by flow cytometry at an emission of 535 nm, and cleaved CCF4 was measured at an emission of 450 nm. Ratios of 450 nm : 535 nm was calculated by dividing the 450 nm MFI by the 535 nm MFI from a single sample. ( C ) DCs were pre-treated with vehicle or chloroquine (CQ) at 1 or 10 µM for 2 hours and then co-cultured with apoptotic B16-OVA cells (5 apoptotic cells: 1 DC). Eighteen hours later, DCs were harvested for flow cytometry analysis of H2-K b -OVA 257-264 expression. ( D ) DCs were pre-treated with vehicle or MG-132 at 1 or 10 µM for 2 hours and then co-cultured in fresh media with apoptotic B16-OVA cells (5 apoptotic cells: 1 DC). Eighteen hours later, DCs were harvested for flow cytometry analysis of H2-K b -OVA 257-264 expression. ( E ) DCs were pre-treated with vehicle or Brefeldin A at 3 µg/ml for 2 hours and then co-cultured in fresh media with apoptotic B16-OVA cells (5 apoptotic cells: 1 DC). Eighteen hours later, DCs were harvested for flow cytometry analysis of H2-K b -OVA 257-264 expression. Data are expressed as mean ± SEM. No less than two independent experiments were performed, with 3-5 replicates per condition. Significance was calculated using 2-way ANOVA (*p
    Figure Legend Snippet: Rubcn -/- DCs exhibit increased phagosome-to-cytosol escape and proteasome-mediated generation of peptides. Bone marrow-derived dendritic cells (DCs) were generated from Rubcn +/+ (black) and Rubcn -/- (red) mice in vitro with FLT3-L for 7 days. ( A-B ) DCs were loaded with 1 µm CCF4, then co-cultured with apoptotic B16-OVA in the absence or presence of β-lactamase (2 mg/ml) for 90 or 180 minutes at 4°C or 37°C. Uncleaved CCF4 was measured by flow cytometry at an emission of 535 nm, and cleaved CCF4 was measured at an emission of 450 nm. Ratios of 450 nm : 535 nm was calculated by dividing the 450 nm MFI by the 535 nm MFI from a single sample. ( C ) DCs were pre-treated with vehicle or chloroquine (CQ) at 1 or 10 µM for 2 hours and then co-cultured with apoptotic B16-OVA cells (5 apoptotic cells: 1 DC). Eighteen hours later, DCs were harvested for flow cytometry analysis of H2-K b -OVA 257-264 expression. ( D ) DCs were pre-treated with vehicle or MG-132 at 1 or 10 µM for 2 hours and then co-cultured in fresh media with apoptotic B16-OVA cells (5 apoptotic cells: 1 DC). Eighteen hours later, DCs were harvested for flow cytometry analysis of H2-K b -OVA 257-264 expression. ( E ) DCs were pre-treated with vehicle or Brefeldin A at 3 µg/ml for 2 hours and then co-cultured in fresh media with apoptotic B16-OVA cells (5 apoptotic cells: 1 DC). Eighteen hours later, DCs were harvested for flow cytometry analysis of H2-K b -OVA 257-264 expression. Data are expressed as mean ± SEM. No less than two independent experiments were performed, with 3-5 replicates per condition. Significance was calculated using 2-way ANOVA (*p

    Techniques Used: Derivative Assay, Generated, Mouse Assay, In Vitro, Cell Culture, Flow Cytometry, Expressing

    18) Product Images from "Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells"

    Article Title: Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells

    Journal: Oncology Research

    doi: 10.3727/096504020X15929939001042

    MG-132 induces cell death in NSCLC cells. (A) H1299 and H441 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 1, and 5 μM) for 48 h. (B) A549 and H460 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 5, and 20 μM) for 48 h. Shown are representative images from three independent experiments. Scale bar: 400 μm. (C, D) Cell viability of H1299 and H441 cells were evaluated by cell counts. (E, F) Cell viability of A549 and H460 cells were evaluated by cell counts. Results are expressed as percent of cell viability normalized to control cells. The bar graphs represent mean with SD from three independent experiments. * p
    Figure Legend Snippet: MG-132 induces cell death in NSCLC cells. (A) H1299 and H441 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 1, and 5 μM) for 48 h. (B) A549 and H460 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 5, and 20 μM) for 48 h. Shown are representative images from three independent experiments. Scale bar: 400 μm. (C, D) Cell viability of H1299 and H441 cells were evaluated by cell counts. (E, F) Cell viability of A549 and H460 cells were evaluated by cell counts. Results are expressed as percent of cell viability normalized to control cells. The bar graphs represent mean with SD from three independent experiments. * p

    Techniques Used: Microscopy

    Proteasome inhibitors reduce c-Met expression in NSCLC cells. (A–D) H1299, H441, A549, and H460 cells were treated with increasing doses of bortezomib (0, 10, 50, and 100 nM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (E–H) H1299 and H441 cells were treated with increasing doses of MG-132 (0, 0.5, 1, 2, and 5 μM) for 48 h, while A549 and H460 cells were treated with higher doses of MG-132 (0, 5, 10, and 20 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (I–L) H1299, H441, A549, and H460 cells were treated with increasing doses of ONX-0914 (0, 1, 2, 5, and 10 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Shown are representative blots from three independent experiments. Intensities of blots were quantified by ImageJ software. Expression of c-Met was normalized to β-actin. The results are shown as mean ± SD of three independent experiments. * p
    Figure Legend Snippet: Proteasome inhibitors reduce c-Met expression in NSCLC cells. (A–D) H1299, H441, A549, and H460 cells were treated with increasing doses of bortezomib (0, 10, 50, and 100 nM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (E–H) H1299 and H441 cells were treated with increasing doses of MG-132 (0, 0.5, 1, 2, and 5 μM) for 48 h, while A549 and H460 cells were treated with higher doses of MG-132 (0, 5, 10, and 20 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (I–L) H1299, H441, A549, and H460 cells were treated with increasing doses of ONX-0914 (0, 1, 2, 5, and 10 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Shown are representative blots from three independent experiments. Intensities of blots were quantified by ImageJ software. Expression of c-Met was normalized to β-actin. The results are shown as mean ± SD of three independent experiments. * p

    Techniques Used: Expressing, Software

    Proteasome inhibitors increase PARP and caspase 3 cleavage and change p53 expression. (A) H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (B) H460 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (C) A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. Shown are representative blots from three independent experiments.
    Figure Legend Snippet: Proteasome inhibitors increase PARP and caspase 3 cleavage and change p53 expression. (A) H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (B) H460 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (C) A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. Shown are representative blots from three independent experiments.

    Techniques Used: Expressing

    The proteasome inhibitors have different effects on c-Met mRNA levels in NSCLC cells. (A–D) NSCLC cells were treated with 100 nM bortezomib for 48 h. c-Met mRNA levels were evaluated by real-time PCR. (E) c-Met mRNA levels in H1299 cells were evaluated after 5 μM MG-132 treatment for 48 h. (F) c-Met mRNA levels in H441 cells were evaluated after 2 μM MG-132 treatment for 48 h. (G) c-Met mRNA levels in H1299 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. (H) c-Met mRNA levels in H441 cells were evaluated after 5 μM ONX 0914 treatment for 48 h. (I) c-Met mRNA levels in H460 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. * p
    Figure Legend Snippet: The proteasome inhibitors have different effects on c-Met mRNA levels in NSCLC cells. (A–D) NSCLC cells were treated with 100 nM bortezomib for 48 h. c-Met mRNA levels were evaluated by real-time PCR. (E) c-Met mRNA levels in H1299 cells were evaluated after 5 μM MG-132 treatment for 48 h. (F) c-Met mRNA levels in H441 cells were evaluated after 2 μM MG-132 treatment for 48 h. (G) c-Met mRNA levels in H1299 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. (H) c-Met mRNA levels in H441 cells were evaluated after 5 μM ONX 0914 treatment for 48 h. (I) c-Met mRNA levels in H460 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. * p

    Techniques Used: Real-time Polymerase Chain Reaction

    Effect of proteasome inhibitors on CHX-mediated c-Met degradation. (A) H441 cells were treated with 20 μg/ml of CHX with or without MG132 (5 μM), bortezomib (100 nM), or ONX 0914 (10 μM). Cells were harvested at each time point over 0–8 h for immune blotting. Data represent one of three independent experiments with similar results. (B) Quantitative analysis of the immunoblots shown in (A) using ImageJ software. The results are shown as mean ± SD of three independent experiments. (C) A549 and H441 cells were transfected with c-Met-V5 plasmids for 1 day, and then were treated with proteasome inhibitors for an additional 48 h. H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM). A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM). Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Cell lysates were analyzed by immunoblotting with antibodies against V5 and β-actin.
    Figure Legend Snippet: Effect of proteasome inhibitors on CHX-mediated c-Met degradation. (A) H441 cells were treated with 20 μg/ml of CHX with or without MG132 (5 μM), bortezomib (100 nM), or ONX 0914 (10 μM). Cells were harvested at each time point over 0–8 h for immune blotting. Data represent one of three independent experiments with similar results. (B) Quantitative analysis of the immunoblots shown in (A) using ImageJ software. The results are shown as mean ± SD of three independent experiments. (C) A549 and H441 cells were transfected with c-Met-V5 plasmids for 1 day, and then were treated with proteasome inhibitors for an additional 48 h. H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM). A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM). Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Cell lysates were analyzed by immunoblotting with antibodies against V5 and β-actin.

    Techniques Used: Western Blot, Software, Transfection

    19) Product Images from "CACYBP Enhances Cytoplasmic Retention of P27Kip1 to Promote Hepatocellular Carcinoma Progression in the Absence of RNF41 Mediated Degradation"

    Article Title: CACYBP Enhances Cytoplasmic Retention of P27Kip1 to Promote Hepatocellular Carcinoma Progression in the Absence of RNF41 Mediated Degradation

    Journal: Theranostics

    doi: 10.7150/thno.36838

    RNF41 promotes the degradation of CACYBP. (A) Expression level of Flag-tagged CACYBP was steadily reduced with increasing amounts of RNF41 in Huh7 and SK-Hep-1 cells. (B) RNF41 with E3 ligase activity reduced the accumulation of exogenously expressed CACYBP, but this did not occur in the presence of the proteasome inhibitor MG132 or the lysosome inhibitors chloroquine or 3-MA. 293T cells were transfected with the indicated plasmids and incubated for 24 h. The cells were then incubated in fresh medium with none, 10 μM MG-132, 50 μM chloroquine, or 5 mM 3-MA for an additional 12 h. The cell lysates were analyzed by immunoblotting using the indicated antibody. Tubulin was used as a loading control. (C) Quantification of the expression level of Flag-tagged CACYBP after normalization to Tubulin. (D) Protein turnover of exogenously expressing Flag-tagged CACYBP in the presence of RNF41 WT or D56V mutant over the course of 8 h following the addition of 10 μg/mL cycloheximide. Tubulin was used as a loading control. (E) Quantification of the percentage of Flag-tagged CACYBP in the presence of RNF41 WT or D56V mutant for each time point compared to 0 h in (D). (F) RNF41 stimulates the poly-ubiquitination of CACYBP in vivo . 293T cells were transfected with the indicated plasmids and incubated for 24 h. Cell lysates were subjected to immunoprecipitation with His affinity nickel beads. Immunoblotting analysis was conducted for the indicated proteins. The red asterisks in lane 3 indicate nonspecific bands. (G) Representative images of RNF41 expression from the same sample slices used for examining CACYBP expression by immunohistochemistry analysis. Scale bar for the left column: 400 μm; Scale bar for the middle and right columns: 100 μm. T: tumor; N: non-tumor. (H) Scatterplot of expression scores of CACYBP vs RNF41 with a regression line showing a negative correlation. (I) RNF41 expression showed no significant association with OS (upper panel) and DFS (lower panel) in the HCC cohort according to Kaplan-Meier analysis. RNF41 high: 76 samples; RNF41 low: 54 samples. *P
    Figure Legend Snippet: RNF41 promotes the degradation of CACYBP. (A) Expression level of Flag-tagged CACYBP was steadily reduced with increasing amounts of RNF41 in Huh7 and SK-Hep-1 cells. (B) RNF41 with E3 ligase activity reduced the accumulation of exogenously expressed CACYBP, but this did not occur in the presence of the proteasome inhibitor MG132 or the lysosome inhibitors chloroquine or 3-MA. 293T cells were transfected with the indicated plasmids and incubated for 24 h. The cells were then incubated in fresh medium with none, 10 μM MG-132, 50 μM chloroquine, or 5 mM 3-MA for an additional 12 h. The cell lysates were analyzed by immunoblotting using the indicated antibody. Tubulin was used as a loading control. (C) Quantification of the expression level of Flag-tagged CACYBP after normalization to Tubulin. (D) Protein turnover of exogenously expressing Flag-tagged CACYBP in the presence of RNF41 WT or D56V mutant over the course of 8 h following the addition of 10 μg/mL cycloheximide. Tubulin was used as a loading control. (E) Quantification of the percentage of Flag-tagged CACYBP in the presence of RNF41 WT or D56V mutant for each time point compared to 0 h in (D). (F) RNF41 stimulates the poly-ubiquitination of CACYBP in vivo . 293T cells were transfected with the indicated plasmids and incubated for 24 h. Cell lysates were subjected to immunoprecipitation with His affinity nickel beads. Immunoblotting analysis was conducted for the indicated proteins. The red asterisks in lane 3 indicate nonspecific bands. (G) Representative images of RNF41 expression from the same sample slices used for examining CACYBP expression by immunohistochemistry analysis. Scale bar for the left column: 400 μm; Scale bar for the middle and right columns: 100 μm. T: tumor; N: non-tumor. (H) Scatterplot of expression scores of CACYBP vs RNF41 with a regression line showing a negative correlation. (I) RNF41 expression showed no significant association with OS (upper panel) and DFS (lower panel) in the HCC cohort according to Kaplan-Meier analysis. RNF41 high: 76 samples; RNF41 low: 54 samples. *P

    Techniques Used: Expressing, Activity Assay, Transfection, Incubation, Mutagenesis, In Vivo, Immunoprecipitation, Immunohistochemistry

    20) Product Images from "Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised"

    Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

    Journal: eLife

    doi: 10.7554/eLife.64977

    Autophagy pathway activity is higher in MAL3-101-resistant cells. ( A ) Chymotrypsin-like proteasome activity was measured by monitoring AMC fluorescence after Suc-LLVY-AMC was added to cell lysates prepared from MAL3-101 sensitive (closed symbols) and resistant (open symbols) cells. Proteasome-independent activity was established by conducting experiments in the presence of MG 132, which was negligible. Data represent averaged net proteasome activity (in pmol substrate hydrolyzed), ± SEM (n=3). ( B–C ) MAL3-101-sensitive (closed symbols) and resistant (open symbols) cell lines were treated with increasing doses of ( B ) CB-5083 (an ERAD inhibitor) or ( C ) bafilomycin (an autophagy inhibitor) for 72 hr, and cell viability was measured. Data represent the means of three independent experiments, ± SEM. ( D–E ) The levels of ( D ) LC3BII and ( E ) the LC3BII:I ratio were determined from data in   Figure 2  and are expressed as the fold change relative to DMSO under the indicated conditions, ± SEM (n≥3 for bafilomycin and n≥4 for CQ). ( F ) LC3BII accumulation under starvation conditions (EBSS) was analyzed in MAL3-101-sensitive (closed symbols) and resistant (open symbols) cells. Black asterisks correspond to the statistical significance between MDA MB 231 cells (closed circle) and MDA MB 453 and MDA MB 361 (open circle and triangle, respectively), and red asterisks represent the statistical significance between MCF7 cells (closed triangle) and MDA MB 453 and MDA MB 361 (open circle and triangle, respectively). Data represent the fold increase relative to DMSO, ± SEM (n=3). * denotes p
    Figure Legend Snippet: Autophagy pathway activity is higher in MAL3-101-resistant cells. ( A ) Chymotrypsin-like proteasome activity was measured by monitoring AMC fluorescence after Suc-LLVY-AMC was added to cell lysates prepared from MAL3-101 sensitive (closed symbols) and resistant (open symbols) cells. Proteasome-independent activity was established by conducting experiments in the presence of MG 132, which was negligible. Data represent averaged net proteasome activity (in pmol substrate hydrolyzed), ± SEM (n=3). ( B–C ) MAL3-101-sensitive (closed symbols) and resistant (open symbols) cell lines were treated with increasing doses of ( B ) CB-5083 (an ERAD inhibitor) or ( C ) bafilomycin (an autophagy inhibitor) for 72 hr, and cell viability was measured. Data represent the means of three independent experiments, ± SEM. ( D–E ) The levels of ( D ) LC3BII and ( E ) the LC3BII:I ratio were determined from data in Figure 2 and are expressed as the fold change relative to DMSO under the indicated conditions, ± SEM (n≥3 for bafilomycin and n≥4 for CQ). ( F ) LC3BII accumulation under starvation conditions (EBSS) was analyzed in MAL3-101-sensitive (closed symbols) and resistant (open symbols) cells. Black asterisks correspond to the statistical significance between MDA MB 231 cells (closed circle) and MDA MB 453 and MDA MB 361 (open circle and triangle, respectively), and red asterisks represent the statistical significance between MCF7 cells (closed triangle) and MDA MB 453 and MDA MB 361 (open circle and triangle, respectively). Data represent the fold increase relative to DMSO, ± SEM (n=3). * denotes p

    Techniques Used: Activity Assay, Fluorescence, Multiple Displacement Amplification

    21) Product Images from "COP9 Signalosome- and 26S Proteasome-dependent Regulation of SCFTIR1 Accumulation inArabidopsis * * S⃞"

    Article Title: COP9 Signalosome- and 26S Proteasome-dependent Regulation of SCFTIR1 Accumulation inArabidopsis * * S⃞

    Journal:

    doi: 10.1074/jbc.M809069200

    Post-translational regulation of SCF TIR1 accumulation in wild-type plants. L er np :: TIR1-HAStrep seedlings were grown in liquid MS for 7 days. A , seedlings were incubated for 6 h with the proteasome inhibitor MG-132 (50 μ m (+) or with DMSO
    Figure Legend Snippet: Post-translational regulation of SCF TIR1 accumulation in wild-type plants. L er np :: TIR1-HAStrep seedlings were grown in liquid MS for 7 days. A , seedlings were incubated for 6 h with the proteasome inhibitor MG-132 (50 μ m (+) or with DMSO

    Techniques Used: Incubation

    22) Product Images from "A Comparative Analysis of Methods to Measure Kinetochore-Microtubule Attachment Stability"

    Article Title: A Comparative Analysis of Methods to Measure Kinetochore-Microtubule Attachment Stability

    Journal: Methods in cell biology

    doi: 10.1016/bs.mcb.2020.01.004

    Calcium Treatment. A) Quantification of spindle intensity for prometaphase U2OS cells treated for 2 hours with 5 μM MG-132 before the addition of DMSO, Taxol, or UMK57 for 1 hour. Cells were permeabilized in calcium buffer for 5 minutes and fixed in glutaraldehyde, as described in section IVA. Whole spindle measurements were made on sum intensity projections of fluorescence z-stacks. Data represent the mean + SEM; n=10 cells per condition; ***p ≤ 0.001; n.s., p ≥ 0.05 using two-tailed t test. B) Representative images (maximum intensity projections) of U2OS cells quantified in (A) fixed and stained for α-tubulin (green) and DNA (blue). Scale bar, 10 μm. C) Quantification of spindle intensity for metaphase U2OS cells treated for 2 hours with 5 μM MG-132 before the addition of DMSO, Taxol, or UMK57 for 1 hour. Calcium treatment assay was performed as described in (A). Whole spindle measurements were made on sum intensity projections of fluorescence z-stacks. Data represent the mean + SEM; n=10 cells per condition; ***p ≤ 0.001; n.s., p ≥ 0.05 using two-tailed t test. D) Representative images (maximum intensity projections) of U2OS cells quantified in (C) fixed and stained for α-tubulin (green) and DNA (blue). Scale bar, 10 μm.
    Figure Legend Snippet: Calcium Treatment. A) Quantification of spindle intensity for prometaphase U2OS cells treated for 2 hours with 5 μM MG-132 before the addition of DMSO, Taxol, or UMK57 for 1 hour. Cells were permeabilized in calcium buffer for 5 minutes and fixed in glutaraldehyde, as described in section IVA. Whole spindle measurements were made on sum intensity projections of fluorescence z-stacks. Data represent the mean + SEM; n=10 cells per condition; ***p ≤ 0.001; n.s., p ≥ 0.05 using two-tailed t test. B) Representative images (maximum intensity projections) of U2OS cells quantified in (A) fixed and stained for α-tubulin (green) and DNA (blue). Scale bar, 10 μm. C) Quantification of spindle intensity for metaphase U2OS cells treated for 2 hours with 5 μM MG-132 before the addition of DMSO, Taxol, or UMK57 for 1 hour. Calcium treatment assay was performed as described in (A). Whole spindle measurements were made on sum intensity projections of fluorescence z-stacks. Data represent the mean + SEM; n=10 cells per condition; ***p ≤ 0.001; n.s., p ≥ 0.05 using two-tailed t test. D) Representative images (maximum intensity projections) of U2OS cells quantified in (C) fixed and stained for α-tubulin (green) and DNA (blue). Scale bar, 10 μm.

    Techniques Used: Fluorescence, Two Tailed Test, Staining

    Differential Sensitivities of k-MTs and non-k-MTs to Low Dose Nocodazole Treatment. A) Average k-MT half-life for DMSO or nocodazole-treated RPE1 cells expressing photoactivatable GFP-α-tubulin. Error bars indicate SEM; n ≥ 10 cells per condition; n.s., p ≥ 0.05 using two-tailed t test. B) Average non-k-MT half-life for DMSO or nocodazole-treated RPE1 cells expressing photoactivatable GFP-α-tubulin. Error bars indicate SEM; n ≥ 10 cells per condition; ***p ≤ 0.001 using two-tailed t test. C) Percentage of photoactivated fluorescence intensity attributable to the fast decay process. Data represent the mean percentage for DMSO or nocodazole-treated cells. Error bars indicate SEM; n ≥ 10 cells per condition; ***p ≤ 0.001 using two-tailed t test. D) ). Whole spindle measurements were made on sum intensity projections of fluorescence z-stacks. Data represent the mean ± SEM; n≥18 cells per condition; ***p ≤ 0.001 using two-tailed t test. E) Quantification of spindle intensity for metaphase RPE1 cells treated with DMSO or low doses of nocodazole for 1 hour. Cells were permeabilized in calcium buffer for 5 minutes and fixed in glutaraldehyde, as described in section IVA. Whole spindle measurements were made on sum intensity projections of fluorescence z-stacks. Data represent the mean ± SEM; n≥16 cells per condition; n.s., p ≥ 0.05 using two-tailed t test. F) Quantification of spindle length in RPE1 cells treated for 1 hour with DMSO or low doses of nocodazole. Data represent mean + SEM; n≥15 cells per condition; **p ≤ 0.01; ***p ≤ 0.001 using two-tailed t test. G) Quantification of IKD as measured by distance between Hec1 staining on sister kinetochores in metaphase RPE1 cells treated with DMSO or low doses of nocodazole for 1 hour. Black bars indicate the mean ± SEM; n≥130 kinetochore pairs and N≥16 cells per condition; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 using two-tailed t test. H) Percentage of metaphase cells with at least one Mad2-positive kinetochore. RPE1 Venus-Mad2 cells treated for 2 hours with 5 μM MG-132 before the addition of DMSO or low doses of nocodazole for 1 hour. Analysis performed on MTSB-extracted cells fixed in 3.5% paraformaldehyde and stained with α-GFP (Abcam 1:1000) to detect Venus-Mad2 (n > 130 cells per condition; ***p
    Figure Legend Snippet: Differential Sensitivities of k-MTs and non-k-MTs to Low Dose Nocodazole Treatment. A) Average k-MT half-life for DMSO or nocodazole-treated RPE1 cells expressing photoactivatable GFP-α-tubulin. Error bars indicate SEM; n ≥ 10 cells per condition; n.s., p ≥ 0.05 using two-tailed t test. B) Average non-k-MT half-life for DMSO or nocodazole-treated RPE1 cells expressing photoactivatable GFP-α-tubulin. Error bars indicate SEM; n ≥ 10 cells per condition; ***p ≤ 0.001 using two-tailed t test. C) Percentage of photoactivated fluorescence intensity attributable to the fast decay process. Data represent the mean percentage for DMSO or nocodazole-treated cells. Error bars indicate SEM; n ≥ 10 cells per condition; ***p ≤ 0.001 using two-tailed t test. D) ). Whole spindle measurements were made on sum intensity projections of fluorescence z-stacks. Data represent the mean ± SEM; n≥18 cells per condition; ***p ≤ 0.001 using two-tailed t test. E) Quantification of spindle intensity for metaphase RPE1 cells treated with DMSO or low doses of nocodazole for 1 hour. Cells were permeabilized in calcium buffer for 5 minutes and fixed in glutaraldehyde, as described in section IVA. Whole spindle measurements were made on sum intensity projections of fluorescence z-stacks. Data represent the mean ± SEM; n≥16 cells per condition; n.s., p ≥ 0.05 using two-tailed t test. F) Quantification of spindle length in RPE1 cells treated for 1 hour with DMSO or low doses of nocodazole. Data represent mean + SEM; n≥15 cells per condition; **p ≤ 0.01; ***p ≤ 0.001 using two-tailed t test. G) Quantification of IKD as measured by distance between Hec1 staining on sister kinetochores in metaphase RPE1 cells treated with DMSO or low doses of nocodazole for 1 hour. Black bars indicate the mean ± SEM; n≥130 kinetochore pairs and N≥16 cells per condition; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 using two-tailed t test. H) Percentage of metaphase cells with at least one Mad2-positive kinetochore. RPE1 Venus-Mad2 cells treated for 2 hours with 5 μM MG-132 before the addition of DMSO or low doses of nocodazole for 1 hour. Analysis performed on MTSB-extracted cells fixed in 3.5% paraformaldehyde and stained with α-GFP (Abcam 1:1000) to detect Venus-Mad2 (n > 130 cells per condition; ***p

    Techniques Used: Expressing, Two Tailed Test, Fluorescence, Staining

    23) Product Images from "Two distinct E3 ligases, SCFFBXL19 and HECW1, degrade thyroid transcription factor 1 in normal thyroid epithelial and follicular thyroid carcinoma cells, respectively"

    Article Title: Two distinct E3 ligases, SCFFBXL19 and HECW1, degrade thyroid transcription factor 1 in normal thyroid epithelial and follicular thyroid carcinoma cells, respectively

    Journal: The FASEB Journal

    doi: 10.1096/fj.201900415R

    PMA induces TTF1 degradation in the ubiquitin-proteasome system in both HTori3 and FTC133 cells. A ) HTori3 cells were treated with MG-132 (20 μM) or leupeptin (100 μM) for 1 h prior to PMA treatment for the indicated incubation time. Cell lysates were analyzed by immunoblotting with TTF1 and β-actin antibodies. TTF1 levels from 3 independent experiments were quantified with ImageJ software. B ) FTC133 cells were treated with MG-132 (20 μM) or leupeptin (100 μM) for 1 h prior to PMA treatment for the indicated incubation time. Cell lysates were analyzed by immunoblotting with TTF1 and β-actin antibodies. TTF1 levels from 3 independent experiments were quantified with ImageJ software. C ) HTori3 cell lysates were subjected to immunoprecipitation with IgG and a TTF1 antibody in a denatured condition followed by a ubiquitin immunoblotting. Input lysates were analyzed by immunoblotting with TTF1 and β-actin antibodies. D ) HTori3 cells were treated with either a vehicle or PMA. Denatured cell lysates were subjected to immunoprecipitation with the ubiquitin antibody followed by TTF1 immunoblotting. Input lysates were analyzed by immunoblotting with TTF1 and β-actin antibodies. All of the blots are representative of 3 independent experiments. E ) FTC133 cells were treated with either a vehicle or PMA. Denatured cell lysates were subject to immunoprecipitation with the ubiquitin antibody followed by TTF1 immunoblotting. Input lysates were analyzed by immunoblotting with TTF1 and β-actin antibodies. All of the blots are representative of 3 independent experiments. ** P
    Figure Legend Snippet: PMA induces TTF1 degradation in the ubiquitin-proteasome system in both HTori3 and FTC133 cells. A ) HTori3 cells were treated with MG-132 (20 μM) or leupeptin (100 μM) for 1 h prior to PMA treatment for the indicated incubation time. Cell lysates were analyzed by immunoblotting with TTF1 and β-actin antibodies. TTF1 levels from 3 independent experiments were quantified with ImageJ software. B ) FTC133 cells were treated with MG-132 (20 μM) or leupeptin (100 μM) for 1 h prior to PMA treatment for the indicated incubation time. Cell lysates were analyzed by immunoblotting with TTF1 and β-actin antibodies. TTF1 levels from 3 independent experiments were quantified with ImageJ software. C ) HTori3 cell lysates were subjected to immunoprecipitation with IgG and a TTF1 antibody in a denatured condition followed by a ubiquitin immunoblotting. Input lysates were analyzed by immunoblotting with TTF1 and β-actin antibodies. D ) HTori3 cells were treated with either a vehicle or PMA. Denatured cell lysates were subjected to immunoprecipitation with the ubiquitin antibody followed by TTF1 immunoblotting. Input lysates were analyzed by immunoblotting with TTF1 and β-actin antibodies. All of the blots are representative of 3 independent experiments. E ) FTC133 cells were treated with either a vehicle or PMA. Denatured cell lysates were subject to immunoprecipitation with the ubiquitin antibody followed by TTF1 immunoblotting. Input lysates were analyzed by immunoblotting with TTF1 and β-actin antibodies. All of the blots are representative of 3 independent experiments. ** P

    Techniques Used: Incubation, Software, Immunoprecipitation

    24) Product Images from "Combination therapy with proteasome inhibitors and TLR agonists enhances tumour cell death and IL-1β production"

    Article Title: Combination therapy with proteasome inhibitors and TLR agonists enhances tumour cell death and IL-1β production

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0194-1

    Proteasome inhibitors induce IL-1β secretion. a THP-1 cells were pre-treated with LPS (10 ng/ml) for 2 h prior to exposure to 5 μM of proteasome inhibitors (MG-132, bortezomib and carfilzomib). Supernatants were collected 1, 8 and 24 h after proteasome inhibition and assayed for IL-1β secretion. b Peripheral blood-derived human macrophages and monocytes were stimulated as in a and assayed for IL-1β secretion after 24 h. c THP-1 and d U937 cells were pre-treated with 10 ng/ml LPS for 2 h prior to proteasome inhibitor treatment. Supernatants were collected and assayed for IL-1β levels after 24 h. n = 3 for all experiments (independent experiments for cell lines, individuals for primary cells). Data were analysed using two-way ANOVA and the Bonferroni post-test. ** and *** indicate P
    Figure Legend Snippet: Proteasome inhibitors induce IL-1β secretion. a THP-1 cells were pre-treated with LPS (10 ng/ml) for 2 h prior to exposure to 5 μM of proteasome inhibitors (MG-132, bortezomib and carfilzomib). Supernatants were collected 1, 8 and 24 h after proteasome inhibition and assayed for IL-1β secretion. b Peripheral blood-derived human macrophages and monocytes were stimulated as in a and assayed for IL-1β secretion after 24 h. c THP-1 and d U937 cells were pre-treated with 10 ng/ml LPS for 2 h prior to proteasome inhibitor treatment. Supernatants were collected and assayed for IL-1β levels after 24 h. n = 3 for all experiments (independent experiments for cell lines, individuals for primary cells). Data were analysed using two-way ANOVA and the Bonferroni post-test. ** and *** indicate P

    Techniques Used: Inhibition, Derivative Assay

    Proteasome inhibition induces processing of bioactive IL-1β. a Conditioned media from THP-1 cells pre-treated 2 h with LPS (10 ng/ml) and then stimulated with MG-132 (5 μM), LPS with vehicle or vehicle only for 24 h, were used to stimulate HEK293 null 1 (NF-κB/AP-1 reporter) cells ( n = 3) in a 1:2 ratio with DMEM. As a control, 2 ng/ml of recombinant IL-1β was used to stimulate these cells in parallel. Responses were measured in the presence of either media or IL-1Ra (200 ng/ml) pre-treatment. b Supernatants from THP-1 cells stimulated as previously indicated for 24 h were blotted for IL-1β processing ( n = 3). c THP-1 supernatants stimulated with LPS or LPS+MG were examined for the appearance of cleaved IL-1β over 24 h ( n = 3). Data were analysed using two-way ANOVA and the Bonferroni post-test. *** indicates P
    Figure Legend Snippet: Proteasome inhibition induces processing of bioactive IL-1β. a Conditioned media from THP-1 cells pre-treated 2 h with LPS (10 ng/ml) and then stimulated with MG-132 (5 μM), LPS with vehicle or vehicle only for 24 h, were used to stimulate HEK293 null 1 (NF-κB/AP-1 reporter) cells ( n = 3) in a 1:2 ratio with DMEM. As a control, 2 ng/ml of recombinant IL-1β was used to stimulate these cells in parallel. Responses were measured in the presence of either media or IL-1Ra (200 ng/ml) pre-treatment. b Supernatants from THP-1 cells stimulated as previously indicated for 24 h were blotted for IL-1β processing ( n = 3). c THP-1 supernatants stimulated with LPS or LPS+MG were examined for the appearance of cleaved IL-1β over 24 h ( n = 3). Data were analysed using two-way ANOVA and the Bonferroni post-test. *** indicates P

    Techniques Used: Inhibition, Recombinant

    IL-1β secretion can occur with priming of various TLRs and correlates with cell death. a THP-1 cells transiently transfected with pro-IL-1β-expressing plasmid were treated with 5 μM MG-132. Lysates and supernatants were harvested at 0, 8, 10 and 24 h post exposure and blotted for IL-1β processing (data are representative of n = 3 experiments). b THP-1 cells were treated with 500 ng/ml flagellin, 100 ng/ml Pam3CSK4, 2.5 µg/ml R848 or 5 μM CpG DNA 2 h prior to proteasome inhibitor treatment for 24 h ( n = 3). c THP-1 X-Blue (NF-κB/AP-1 reporter) cells were examined for the ability of TLR ligands to activate NF-κB/AP-1 activity over 24 h ( n = 3–5). d LPS (10 ng/ml), flagellin, R848 and CpG-treated THP-1 cells (2 h) were exposed to 2 nM, 100 nM or 5 μM bortezomib and checked for viability after 24 h via MTS assay ( n = 3). d Data were analysed using one-way or two-way ANOVA with Bonferroni post-test. *** indicates P
    Figure Legend Snippet: IL-1β secretion can occur with priming of various TLRs and correlates with cell death. a THP-1 cells transiently transfected with pro-IL-1β-expressing plasmid were treated with 5 μM MG-132. Lysates and supernatants were harvested at 0, 8, 10 and 24 h post exposure and blotted for IL-1β processing (data are representative of n = 3 experiments). b THP-1 cells were treated with 500 ng/ml flagellin, 100 ng/ml Pam3CSK4, 2.5 µg/ml R848 or 5 μM CpG DNA 2 h prior to proteasome inhibitor treatment for 24 h ( n = 3). c THP-1 X-Blue (NF-κB/AP-1 reporter) cells were examined for the ability of TLR ligands to activate NF-κB/AP-1 activity over 24 h ( n = 3–5). d LPS (10 ng/ml), flagellin, R848 and CpG-treated THP-1 cells (2 h) were exposed to 2 nM, 100 nM or 5 μM bortezomib and checked for viability after 24 h via MTS assay ( n = 3). d Data were analysed using one-way or two-way ANOVA with Bonferroni post-test. *** indicates P

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Activity Assay, MTS Assay

    Divergent effects of proteasome inhibition on IL-1β synthesis, degradation and secretion are dependent on relative treatment order. THP-1 cells were exposed to the ER stressors tunicamycin (10 μg/ml), thapsigargin (10 μM) and MG-132 (5 μM) either 2 h a before or b after stimulation with LPS (10 ng/ml). c MG-132, bortezomib and carfilzomib (all 5 μM) were added to THP-1 cultures either before (2 h, 1 h), at the same time or 1 h after LPS stimulation (time = 0). Supernatants were harvested 24 h after stimulation. d Proteasome inhibitors (5 μM) were applied 2 h prior to LPS stimulation (10 ng/ml) and harvesting of cell lysates occuring at 0.5, 1.5 and 3 h post stimulation. Lysates were blotted for pro-IL-1β and quantified for its presence relative to LPS + DMSO. e THP-1 X Blue (NF-κB/AP-1 reporter) cells were treated as in d , but supernatants were collected after 6 h and assayed for SEAP activity overnight. f THP-1 cells were stimulated with LPS (1 ng/ml) for 4 h prior to removal of medium and treatment with bortezomib (5 μM) and/or cycloheximide (200 μg/ml). Lysates were collected at 0, 1.5, 3 and 5 h and blotted for pro-IL-1β to observe its relative degradation. Data were analysed using two-way ANOVA and the Bonferroni post-test. *, ** and *** indicate P
    Figure Legend Snippet: Divergent effects of proteasome inhibition on IL-1β synthesis, degradation and secretion are dependent on relative treatment order. THP-1 cells were exposed to the ER stressors tunicamycin (10 μg/ml), thapsigargin (10 μM) and MG-132 (5 μM) either 2 h a before or b after stimulation with LPS (10 ng/ml). c MG-132, bortezomib and carfilzomib (all 5 μM) were added to THP-1 cultures either before (2 h, 1 h), at the same time or 1 h after LPS stimulation (time = 0). Supernatants were harvested 24 h after stimulation. d Proteasome inhibitors (5 μM) were applied 2 h prior to LPS stimulation (10 ng/ml) and harvesting of cell lysates occuring at 0.5, 1.5 and 3 h post stimulation. Lysates were blotted for pro-IL-1β and quantified for its presence relative to LPS + DMSO. e THP-1 X Blue (NF-κB/AP-1 reporter) cells were treated as in d , but supernatants were collected after 6 h and assayed for SEAP activity overnight. f THP-1 cells were stimulated with LPS (1 ng/ml) for 4 h prior to removal of medium and treatment with bortezomib (5 μM) and/or cycloheximide (200 μg/ml). Lysates were collected at 0, 1.5, 3 and 5 h and blotted for pro-IL-1β to observe its relative degradation. Data were analysed using two-way ANOVA and the Bonferroni post-test. *, ** and *** indicate P

    Techniques Used: Inhibition, Activity Assay

    25) Product Images from "A novel orally available seleno-purine molecule suppresses triple-negative breast cancer cell proliferation and progression to metastasis by inducing cytostatic autophagy"

    Article Title: A novel orally available seleno-purine molecule suppresses triple-negative breast cancer cell proliferation and progression to metastasis by inducing cytostatic autophagy

    Journal: Autophagy

    doi: 10.1080/15548627.2019.1582951

    SLLN-15 induced autophagy through downregulation of AURKA-AKT-MTOR axis. ( a ) MDA-MB-231 and BT-20 cells were treated with either DMSO or indicated concentration of SLLN-15 for 24 h, lysed, immunoblotted with antibodies against AURKA/B/C, AURKA, AURKB and GAPDH (internal control). ( b ) Lysates from cells treated with either DMSO or 10 μM of SLLN-15 alone or pretreated with MG-132 (2 μM, 24 h) for 24 h were immunoblotted with antibodies against p-AURKA/B, AURKA, AURKB and GAPDH (internal control). (c) BT-20 cells were treated with either DMSO or 10 μM of SLLN-15 for the indicated time, lysed and immunoblotted with anti-p-AURKA/B, anti-AURKA, anti-AURKB, LC3B and GAPDH (internal control) antibodies. ( d ) 293T cells were transfected with an empty vector (pCMV), pCMV- AURKA or pCMV- AURKB plasmids for 48 h, and then cells were treated with 10 μM of SLLN-15 for another 24 h and immunoblotted with antibodies against FLAG-tag, LC3B, p-S240/244-RPS6, p-S473-AKT, p-T421/S424-RPS6KB and anti-GAPDH as an internal control. (e) 293T cells transfected with an empty vector (pCMV), pCMV- AURKA or pCMV- AURKB plasmids for 48 h were then treated with either DMSO or SLLN-15 (10 μM) for 24 h, fixed and stained with anti-LC3 antibody (green) and with DAPI (blue); scale bar: 20 μm (left); quantification on the right shows the average number of LC3 puncta per cell (mean ± SEM, n = 30 cells from 3 independent experiments, *p
    Figure Legend Snippet: SLLN-15 induced autophagy through downregulation of AURKA-AKT-MTOR axis. ( a ) MDA-MB-231 and BT-20 cells were treated with either DMSO or indicated concentration of SLLN-15 for 24 h, lysed, immunoblotted with antibodies against AURKA/B/C, AURKA, AURKB and GAPDH (internal control). ( b ) Lysates from cells treated with either DMSO or 10 μM of SLLN-15 alone or pretreated with MG-132 (2 μM, 24 h) for 24 h were immunoblotted with antibodies against p-AURKA/B, AURKA, AURKB and GAPDH (internal control). (c) BT-20 cells were treated with either DMSO or 10 μM of SLLN-15 for the indicated time, lysed and immunoblotted with anti-p-AURKA/B, anti-AURKA, anti-AURKB, LC3B and GAPDH (internal control) antibodies. ( d ) 293T cells were transfected with an empty vector (pCMV), pCMV- AURKA or pCMV- AURKB plasmids for 48 h, and then cells were treated with 10 μM of SLLN-15 for another 24 h and immunoblotted with antibodies against FLAG-tag, LC3B, p-S240/244-RPS6, p-S473-AKT, p-T421/S424-RPS6KB and anti-GAPDH as an internal control. (e) 293T cells transfected with an empty vector (pCMV), pCMV- AURKA or pCMV- AURKB plasmids for 48 h were then treated with either DMSO or SLLN-15 (10 μM) for 24 h, fixed and stained with anti-LC3 antibody (green) and with DAPI (blue); scale bar: 20 μm (left); quantification on the right shows the average number of LC3 puncta per cell (mean ± SEM, n = 30 cells from 3 independent experiments, *p

    Techniques Used: Multiple Displacement Amplification, Concentration Assay, Transfection, Plasmid Preparation, FLAG-tag, Staining

    26) Product Images from "Role of Ubiquitin and Proteasomes in Phagosome Maturation"

    Article Title: Role of Ubiquitin and Proteasomes in Phagosome Maturation

    Journal:

    doi: 10.1091/mbc.E04-06-0464

    Proteasome inhibition prevents loss of FcγRIIA from the phagosome. CHO cells stably transfected with FcγRIIA-GFP were either left untreated (A) or pretreated with 10 μM MG-132 for 3 h (C) before phagocytosis of opsonized beads
    Figure Legend Snippet: Proteasome inhibition prevents loss of FcγRIIA from the phagosome. CHO cells stably transfected with FcγRIIA-GFP were either left untreated (A) or pretreated with 10 μM MG-132 for 3 h (C) before phagocytosis of opsonized beads

    Techniques Used: Inhibition, Stable Transfection, Transfection

    Proteasome inhibition has no effect on fusion of phagosomes with MVB or lysosomes. RAW macrophages were either left untreated (A, D, and G) or were pretreated with 10 μM MG-132 for 1.5 h (B, E, and H) and allowed to ingest opsonized beads for
    Figure Legend Snippet: Proteasome inhibition has no effect on fusion of phagosomes with MVB or lysosomes. RAW macrophages were either left untreated (A, D, and G) or were pretreated with 10 μM MG-132 for 1.5 h (B, E, and H) and allowed to ingest opsonized beads for

    Techniques Used: Inhibition

    The effects of proteasome inhibition on fission are not through depletion of cellular free ubiquitin. (A) RAW macrophages were either left untreated (control) or were pretreated with MG-132 for 1.5 h and then allowed to ingest opsonized beads for 12 min
    Figure Legend Snippet: The effects of proteasome inhibition on fission are not through depletion of cellular free ubiquitin. (A) RAW macrophages were either left untreated (control) or were pretreated with MG-132 for 1.5 h and then allowed to ingest opsonized beads for 12 min

    Techniques Used: Inhibition

    Translocation of the cytosolic tail of FcγRIIA to an acidic compartment is prevented by proteasome inhibitors. CHO cells stably transfected with FcγRIIA-GFP were pretreated with 10 μM MG-132 for 3 h and allowed to ingest opsonized
    Figure Legend Snippet: Translocation of the cytosolic tail of FcγRIIA to an acidic compartment is prevented by proteasome inhibitors. CHO cells stably transfected with FcγRIIA-GFP were pretreated with 10 μM MG-132 for 3 h and allowed to ingest opsonized

    Techniques Used: Translocation Assay, Stable Transfection, Transfection

    27) Product Images from "Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells"

    Article Title: Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells

    Journal: Oncology Research

    doi: 10.3727/096504020X15929939001042

    MG-132 induces cell death in NSCLC cells. (A) H1299 and H441 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 1, and 5 μM) for 48 h. (B) A549 and H460 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 5, and 20 μM) for 48 h. Shown are representative images from three independent experiments. Scale bar: 400 μm. (C, D) Cell viability of H1299 and H441 cells were evaluated by cell counts. (E, F) Cell viability of A549 and H460 cells were evaluated by cell counts. Results are expressed as percent of cell viability normalized to control cells. The bar graphs represent mean with SD from three independent experiments. * p
    Figure Legend Snippet: MG-132 induces cell death in NSCLC cells. (A) H1299 and H441 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 1, and 5 μM) for 48 h. (B) A549 and H460 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 5, and 20 μM) for 48 h. Shown are representative images from three independent experiments. Scale bar: 400 μm. (C, D) Cell viability of H1299 and H441 cells were evaluated by cell counts. (E, F) Cell viability of A549 and H460 cells were evaluated by cell counts. Results are expressed as percent of cell viability normalized to control cells. The bar graphs represent mean with SD from three independent experiments. * p

    Techniques Used: Microscopy

    Proteasome inhibitors reduce c-Met expression in NSCLC cells. (A–D) H1299, H441, A549, and H460 cells were treated with increasing doses of bortezomib (0, 10, 50, and 100 nM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (E–H) H1299 and H441 cells were treated with increasing doses of MG-132 (0, 0.5, 1, 2, and 5 μM) for 48 h, while A549 and H460 cells were treated with higher doses of MG-132 (0, 5, 10, and 20 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (I–L) H1299, H441, A549, and H460 cells were treated with increasing doses of ONX-0914 (0, 1, 2, 5, and 10 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Shown are representative blots from three independent experiments. Intensities of blots were quantified by ImageJ software. Expression of c-Met was normalized to β-actin. The results are shown as mean ± SD of three independent experiments. * p
    Figure Legend Snippet: Proteasome inhibitors reduce c-Met expression in NSCLC cells. (A–D) H1299, H441, A549, and H460 cells were treated with increasing doses of bortezomib (0, 10, 50, and 100 nM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (E–H) H1299 and H441 cells were treated with increasing doses of MG-132 (0, 0.5, 1, 2, and 5 μM) for 48 h, while A549 and H460 cells were treated with higher doses of MG-132 (0, 5, 10, and 20 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (I–L) H1299, H441, A549, and H460 cells were treated with increasing doses of ONX-0914 (0, 1, 2, 5, and 10 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Shown are representative blots from three independent experiments. Intensities of blots were quantified by ImageJ software. Expression of c-Met was normalized to β-actin. The results are shown as mean ± SD of three independent experiments. * p

    Techniques Used: Expressing, Software

    Proteasome inhibitors increase PARP and caspase 3 cleavage and change p53 expression. (A) H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (B) H460 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (C) A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. Shown are representative blots from three independent experiments.
    Figure Legend Snippet: Proteasome inhibitors increase PARP and caspase 3 cleavage and change p53 expression. (A) H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (B) H460 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (C) A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. Shown are representative blots from three independent experiments.

    Techniques Used: Expressing

    The proteasome inhibitors have different effects on c-Met mRNA levels in NSCLC cells. (A–D) NSCLC cells were treated with 100 nM bortezomib for 48 h. c-Met mRNA levels were evaluated by real-time PCR. (E) c-Met mRNA levels in H1299 cells were evaluated after 5 μM MG-132 treatment for 48 h. (F) c-Met mRNA levels in H441 cells were evaluated after 2 μM MG-132 treatment for 48 h. (G) c-Met mRNA levels in H1299 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. (H) c-Met mRNA levels in H441 cells were evaluated after 5 μM ONX 0914 treatment for 48 h. (I) c-Met mRNA levels in H460 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. * p
    Figure Legend Snippet: The proteasome inhibitors have different effects on c-Met mRNA levels in NSCLC cells. (A–D) NSCLC cells were treated with 100 nM bortezomib for 48 h. c-Met mRNA levels were evaluated by real-time PCR. (E) c-Met mRNA levels in H1299 cells were evaluated after 5 μM MG-132 treatment for 48 h. (F) c-Met mRNA levels in H441 cells were evaluated after 2 μM MG-132 treatment for 48 h. (G) c-Met mRNA levels in H1299 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. (H) c-Met mRNA levels in H441 cells were evaluated after 5 μM ONX 0914 treatment for 48 h. (I) c-Met mRNA levels in H460 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. * p

    Techniques Used: Real-time Polymerase Chain Reaction

    Effect of proteasome inhibitors on CHX-mediated c-Met degradation. (A) H441 cells were treated with 20 μg/ml of CHX with or without MG132 (5 μM), bortezomib (100 nM), or ONX 0914 (10 μM). Cells were harvested at each time point over 0–8 h for immune blotting. Data represent one of three independent experiments with similar results. (B) Quantitative analysis of the immunoblots shown in (A) using ImageJ software. The results are shown as mean ± SD of three independent experiments. (C) A549 and H441 cells were transfected with c-Met-V5 plasmids for 1 day, and then were treated with proteasome inhibitors for an additional 48 h. H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM). A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM). Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Cell lysates were analyzed by immunoblotting with antibodies against V5 and β-actin.
    Figure Legend Snippet: Effect of proteasome inhibitors on CHX-mediated c-Met degradation. (A) H441 cells were treated with 20 μg/ml of CHX with or without MG132 (5 μM), bortezomib (100 nM), or ONX 0914 (10 μM). Cells were harvested at each time point over 0–8 h for immune blotting. Data represent one of three independent experiments with similar results. (B) Quantitative analysis of the immunoblots shown in (A) using ImageJ software. The results are shown as mean ± SD of three independent experiments. (C) A549 and H441 cells were transfected with c-Met-V5 plasmids for 1 day, and then were treated with proteasome inhibitors for an additional 48 h. H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM). A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM). Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Cell lysates were analyzed by immunoblotting with antibodies against V5 and β-actin.

    Techniques Used: Western Blot, Software, Transfection

    28) Product Images from "Hsp90B enhances MAST1-mediated cisplatin resistance by protecting MAST1 from proteosomal degradation"

    Article Title: Hsp90B enhances MAST1-mediated cisplatin resistance by protecting MAST1 from proteosomal degradation

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI125963

    CHIP ubiquitinates and degrades MAST1 when unmasked by hsp90B. ( A ) MAST1 interacts with CHIP in cells. GST-pull-down samples from 293T were applied to LC-MS/MS. Spectral counts of CHIP and MAST1 in samples treated with or without 17-AAG are shown. ( B ) Overexpressed CHIP and MAST1 interact in cancer cells. Effect of CHIP overexpression ( C ) or knockout ( D ) on MAST1 levels. ( E ) Comparison of MAST1 protein stability in cells with CHIP modulation was achieved by cycloheximide (CHX) chase assay. Cells with CHIP knockdown were transfected with shRNA-resistant CHIP variants followed by 5 μg/mL CHX treatment for the indicated time. MAST1 amount was determined by density analysis. Representative data are shown. ( F ) In vitro CHIP ubiquitination assay using purified MAST1 WT or 2KR. ( G ) Ubiquitination of MAST1 WT and 2KR in cells. GST pull-down samples from A549 cisR cells treated with MG-132 (10 μM) were immunoblotted with anti-ubiquitin antibody. ( H ) Interaction of MAST1 WT or 2KR, hsp90B, and CHIP in the presence or absence of 17-AAG. MAST1 WT and 2KR protein stability in the absence ( I ) and presence ( J ) of 17-AAG was determined by cycloheximide chase assay. Data shown are representative of 3 ( B – E , I , and J ) and 2 ( F , G , H ) independent biological experiments. Data are mean ± SD from 3 technical replicates. Statistical analysis was performed by 2-way ANOVA for E and 1-way ANOVA for I and J . *** P
    Figure Legend Snippet: CHIP ubiquitinates and degrades MAST1 when unmasked by hsp90B. ( A ) MAST1 interacts with CHIP in cells. GST-pull-down samples from 293T were applied to LC-MS/MS. Spectral counts of CHIP and MAST1 in samples treated with or without 17-AAG are shown. ( B ) Overexpressed CHIP and MAST1 interact in cancer cells. Effect of CHIP overexpression ( C ) or knockout ( D ) on MAST1 levels. ( E ) Comparison of MAST1 protein stability in cells with CHIP modulation was achieved by cycloheximide (CHX) chase assay. Cells with CHIP knockdown were transfected with shRNA-resistant CHIP variants followed by 5 μg/mL CHX treatment for the indicated time. MAST1 amount was determined by density analysis. Representative data are shown. ( F ) In vitro CHIP ubiquitination assay using purified MAST1 WT or 2KR. ( G ) Ubiquitination of MAST1 WT and 2KR in cells. GST pull-down samples from A549 cisR cells treated with MG-132 (10 μM) were immunoblotted with anti-ubiquitin antibody. ( H ) Interaction of MAST1 WT or 2KR, hsp90B, and CHIP in the presence or absence of 17-AAG. MAST1 WT and 2KR protein stability in the absence ( I ) and presence ( J ) of 17-AAG was determined by cycloheximide chase assay. Data shown are representative of 3 ( B – E , I , and J ) and 2 ( F , G , H ) independent biological experiments. Data are mean ± SD from 3 technical replicates. Statistical analysis was performed by 2-way ANOVA for E and 1-way ANOVA for I and J . *** P

    Techniques Used: Chromatin Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Over Expression, Knock-Out, Transfection, shRNA, In Vitro, Ubiquitin Assay, Purification

    Inhibition of hsp90 induces ubiquitination of MAST1 at lysine 317/545 that leads to proteasomal degradation. ( A ) Effect of 17-AAG on MAST1 ubiquitination. 293T cells with GST-MAST1 and HA-tagged ubiquitin (Ub) were treated with 17-AAG for 4 hours and subjected to GST pull down. Anti-HA antibody was used to detect ubiquitinated MAST1. ( B and C ) Effect of 17-AAG on MAST1 proteasomal degradation. Cells were treated with or without MG-132 (10 μM) before addition of 17-AAG (1 μM) in 293T ( B ) or cisplatin-resistant cancer cells ( C ), and exogenous or endogenous MAST1 levels were detected, respectively. cRaf and AKT levels are shown for comparison. ( D ) Effect of 17-AAG on MAST1 ubiquitination and proteasomal degradation in cisplatin-resistant cancer cells. ( E ) MS spectra of ubiquitinated peptide fragments of MAST1. 293T cells with GST-MAST1 were treated with 1 μM of 17-AAG for 4 hours. Ubiquitination at K317 and K545 in MAST1 was identified using LC/MS-MS. ( F ) Ubiquitination of MAST1 WT and K317R or/and K545R mutants upon 17-AAG treatment. ( G ) Degradation of MAST1 WT and K317R/K545R (2KR) upon 17-AAG treatment in KB-3-1 cisR and A549 cisR cells. MAST1 knockdown cells were transfected with shRNA-resistant MAST1 WT or 2KR and treated with indicated concentrations of 17-AAG for 24 hours. Data shown are representative of 2 ( A , B , and F ) and 3 ( C , D , and G ) independent biological experiments.
    Figure Legend Snippet: Inhibition of hsp90 induces ubiquitination of MAST1 at lysine 317/545 that leads to proteasomal degradation. ( A ) Effect of 17-AAG on MAST1 ubiquitination. 293T cells with GST-MAST1 and HA-tagged ubiquitin (Ub) were treated with 17-AAG for 4 hours and subjected to GST pull down. Anti-HA antibody was used to detect ubiquitinated MAST1. ( B and C ) Effect of 17-AAG on MAST1 proteasomal degradation. Cells were treated with or without MG-132 (10 μM) before addition of 17-AAG (1 μM) in 293T ( B ) or cisplatin-resistant cancer cells ( C ), and exogenous or endogenous MAST1 levels were detected, respectively. cRaf and AKT levels are shown for comparison. ( D ) Effect of 17-AAG on MAST1 ubiquitination and proteasomal degradation in cisplatin-resistant cancer cells. ( E ) MS spectra of ubiquitinated peptide fragments of MAST1. 293T cells with GST-MAST1 were treated with 1 μM of 17-AAG for 4 hours. Ubiquitination at K317 and K545 in MAST1 was identified using LC/MS-MS. ( F ) Ubiquitination of MAST1 WT and K317R or/and K545R mutants upon 17-AAG treatment. ( G ) Degradation of MAST1 WT and K317R/K545R (2KR) upon 17-AAG treatment in KB-3-1 cisR and A549 cisR cells. MAST1 knockdown cells were transfected with shRNA-resistant MAST1 WT or 2KR and treated with indicated concentrations of 17-AAG for 24 hours. Data shown are representative of 2 ( A , B , and F ) and 3 ( C , D , and G ) independent biological experiments.

    Techniques Used: Inhibition, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Transfection, shRNA

    29) Product Images from "Bortezomib induces apoptosis via Bim and Bik up-regulation and synergizes with cisplatin in the killing of head and neck squamous cell carcinoma cells"

    Article Title: Bortezomib induces apoptosis via Bim and Bik up-regulation and synergizes with cisplatin in the killing of head and neck squamous cell carcinoma cells

    Journal:

    doi: 10.1158/1535-7163.MCT-07-2444

    Induction of HNSCC cell death by proteasome inhibitors. UM-22A, UM-22B, and 1483 cells were left untreated or were treated for 48 h with 0.1% DMSO or varying concentrations of MG-132 ( A ) or bortezomib ( B ). Following treatment, MTS assays were done. Points
    Figure Legend Snippet: Induction of HNSCC cell death by proteasome inhibitors. UM-22A, UM-22B, and 1483 cells were left untreated or were treated for 48 h with 0.1% DMSO or varying concentrations of MG-132 ( A ) or bortezomib ( B ). Following treatment, MTS assays were done. Points

    Techniques Used:

    30) Product Images from "Role of Ubiquitin and Proteasomes in Phagosome Maturation"

    Article Title: Role of Ubiquitin and Proteasomes in Phagosome Maturation

    Journal:

    doi: 10.1091/mbc.E04-06-0464

    Proteasome inhibition prevents loss of FcγRIIA from the phagosome. CHO cells stably transfected with FcγRIIA-GFP were either left untreated (A) or pretreated with 10 μM MG-132 for 3 h (C) before phagocytosis of opsonized beads
    Figure Legend Snippet: Proteasome inhibition prevents loss of FcγRIIA from the phagosome. CHO cells stably transfected with FcγRIIA-GFP were either left untreated (A) or pretreated with 10 μM MG-132 for 3 h (C) before phagocytosis of opsonized beads

    Techniques Used: Inhibition, Stable Transfection, Transfection

    Proteasome inhibition has no effect on fusion of phagosomes with MVB or lysosomes. RAW macrophages were either left untreated (A, D, and G) or were pretreated with 10 μM MG-132 for 1.5 h (B, E, and H) and allowed to ingest opsonized beads for
    Figure Legend Snippet: Proteasome inhibition has no effect on fusion of phagosomes with MVB or lysosomes. RAW macrophages were either left untreated (A, D, and G) or were pretreated with 10 μM MG-132 for 1.5 h (B, E, and H) and allowed to ingest opsonized beads for

    Techniques Used: Inhibition

    The effects of proteasome inhibition on fission are not through depletion of cellular free ubiquitin. (A) RAW macrophages were either left untreated (control) or were pretreated with MG-132 for 1.5 h and then allowed to ingest opsonized beads for 12 min
    Figure Legend Snippet: The effects of proteasome inhibition on fission are not through depletion of cellular free ubiquitin. (A) RAW macrophages were either left untreated (control) or were pretreated with MG-132 for 1.5 h and then allowed to ingest opsonized beads for 12 min

    Techniques Used: Inhibition

    Translocation of the cytosolic tail of FcγRIIA to an acidic compartment is prevented by proteasome inhibitors. CHO cells stably transfected with FcγRIIA-GFP were pretreated with 10 μM MG-132 for 3 h and allowed to ingest opsonized
    Figure Legend Snippet: Translocation of the cytosolic tail of FcγRIIA to an acidic compartment is prevented by proteasome inhibitors. CHO cells stably transfected with FcγRIIA-GFP were pretreated with 10 μM MG-132 for 3 h and allowed to ingest opsonized

    Techniques Used: Translocation Assay, Stable Transfection, Transfection

    31) Product Images from "COP9 Signalosome- and 26S Proteasome-dependent Regulation of SCFTIR1 Accumulation inArabidopsis * * S⃞"

    Article Title: COP9 Signalosome- and 26S Proteasome-dependent Regulation of SCFTIR1 Accumulation inArabidopsis * * S⃞

    Journal:

    doi: 10.1074/jbc.M809069200

    Post-translational regulation of SCF TIR1 accumulation in wild-type plants. L er np :: TIR1-HAStrep seedlings were grown in liquid MS for 7 days. A , seedlings were incubated for 6 h with the proteasome inhibitor MG-132 (50 μ m (+) or with DMSO
    Figure Legend Snippet: Post-translational regulation of SCF TIR1 accumulation in wild-type plants. L er np :: TIR1-HAStrep seedlings were grown in liquid MS for 7 days. A , seedlings were incubated for 6 h with the proteasome inhibitor MG-132 (50 μ m (+) or with DMSO

    Techniques Used: Incubation

    32) Product Images from "The Function of FK506-Binding Protein 13 in Protein Quality Control Protects Plasma Cells from Endoplasmic Reticulum Stress-Associated Apoptosis"

    Article Title: The Function of FK506-Binding Protein 13 in Protein Quality Control Protects Plasma Cells from Endoplasmic Reticulum Stress-Associated Apoptosis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00222

    FK506-binding protein 13 (FKBP13) promotes ubiquitination-mediated degradation of immunoglobin (Ig) molecules . (A) J558 cells were lysed, immunoprecipitated with anti-goat IgG, anti-FKBP13, or anti-IgA antibodies, and assayed by western blotting. (B–G) J558 cells were transfected with MigR1, MigR1-myc-FKBP13 (FKBP13), pGFP-V-RS-shFKBP13 (shFKBP13), or pGFP-V-RS-SCR (SCR) as indicated. (B) The transfectants were incubated with 1 μM MG-132 or DMSO for 24 h followed by western blotting. (C) Lysates from the transfectants were immnoprecipitated with anti-IgA antibodies and assayed by western blotting. (D,E) GFP + transfectants were sorted by FACS and cultured for 24 h, and supernatant IgA was assayed by enzyme-linked immunosorbent assays. (F) IgA Cα transcripts in the transfectants were assayed by quantitative RT-PCR. (G) J558 cells were transfected with MigR1 or MigR1-myc-FKBP13 constructs together with reporter constructs (pGL3b-UPRE and pRL-CMV), and dual luciferase activities were measured. Firefly luciferase activity was normalized by the Renilla luciferase activity and is shown as relative luciferase units (RLU). All data are representative of at least three independent experiments. * p
    Figure Legend Snippet: FK506-binding protein 13 (FKBP13) promotes ubiquitination-mediated degradation of immunoglobin (Ig) molecules . (A) J558 cells were lysed, immunoprecipitated with anti-goat IgG, anti-FKBP13, or anti-IgA antibodies, and assayed by western blotting. (B–G) J558 cells were transfected with MigR1, MigR1-myc-FKBP13 (FKBP13), pGFP-V-RS-shFKBP13 (shFKBP13), or pGFP-V-RS-SCR (SCR) as indicated. (B) The transfectants were incubated with 1 μM MG-132 or DMSO for 24 h followed by western blotting. (C) Lysates from the transfectants were immnoprecipitated with anti-IgA antibodies and assayed by western blotting. (D,E) GFP + transfectants were sorted by FACS and cultured for 24 h, and supernatant IgA was assayed by enzyme-linked immunosorbent assays. (F) IgA Cα transcripts in the transfectants were assayed by quantitative RT-PCR. (G) J558 cells were transfected with MigR1 or MigR1-myc-FKBP13 constructs together with reporter constructs (pGL3b-UPRE and pRL-CMV), and dual luciferase activities were measured. Firefly luciferase activity was normalized by the Renilla luciferase activity and is shown as relative luciferase units (RLU). All data are representative of at least three independent experiments. * p

    Techniques Used: Binding Assay, Immunoprecipitation, Western Blot, Transfection, Incubation, FACS, Cell Culture, Quantitative RT-PCR, Construct, Luciferase, Activity Assay

    33) Product Images from "Ubiquitin ligases HUWE1 and NEDD4 cooperatively control signal-dependent PRC2-Ezh1α/β-mediated adaptive stress response pathway in skeletal muscle cells"

    Article Title: Ubiquitin ligases HUWE1 and NEDD4 cooperatively control signal-dependent PRC2-Ezh1α/β-mediated adaptive stress response pathway in skeletal muscle cells

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-019-0322-5

    Ezh1β Serine 560 phosphorylation is required for interaction between Ezh1β and NEDD4. a Poly-ubiquitination profiles of Ezh1β-T7, Ezh1βS560A-T7 and Ezh1βS560D-T7 under normal and oxidative stress conditions. HA-ubiquitin/Ezh1β-T7, HA-ubiquitin/Ezh1βS560A-T7 and HA-ubiquitin/Ezh1βS560D-T7 indicate C2C12 cell lines co-expressing HA-Ubiquitin and different forms of Ezh1β-T7 fusion proteins. Total proteins were extracted from these indicated stable cell lines. Ubiquitinated total proteins and ubiquitinated different Ezh1β forms were immunoprecipitated with HA agarose beads. Samples were eluted with HA peptide and running SDS-PAGE, anti-HA and anti-Ezh1β were used to detect total ubiquitinated proteins and ubiquitinated Ezh1β or mutant Ezh1β forms. 100 μM MG-132 was added and treated for 4 h before protein extraction. Ponceau S staining was used as loading control. b Interaction between NEDD4 and different forms of Ezh1β: Ezh1β, Ezh1βS560A and Ezh1βS560D. Cytosolic proteins were extracted and tandemly immunoprecipitated with Flag and HA agarose beads. NEDD4 antibody was used for immunoblotting analysis. Ponceau S staining was used as loading control
    Figure Legend Snippet: Ezh1β Serine 560 phosphorylation is required for interaction between Ezh1β and NEDD4. a Poly-ubiquitination profiles of Ezh1β-T7, Ezh1βS560A-T7 and Ezh1βS560D-T7 under normal and oxidative stress conditions. HA-ubiquitin/Ezh1β-T7, HA-ubiquitin/Ezh1βS560A-T7 and HA-ubiquitin/Ezh1βS560D-T7 indicate C2C12 cell lines co-expressing HA-Ubiquitin and different forms of Ezh1β-T7 fusion proteins. Total proteins were extracted from these indicated stable cell lines. Ubiquitinated total proteins and ubiquitinated different Ezh1β forms were immunoprecipitated with HA agarose beads. Samples were eluted with HA peptide and running SDS-PAGE, anti-HA and anti-Ezh1β were used to detect total ubiquitinated proteins and ubiquitinated Ezh1β or mutant Ezh1β forms. 100 μM MG-132 was added and treated for 4 h before protein extraction. Ponceau S staining was used as loading control. b Interaction between NEDD4 and different forms of Ezh1β: Ezh1β, Ezh1βS560A and Ezh1βS560D. Cytosolic proteins were extracted and tandemly immunoprecipitated with Flag and HA agarose beads. NEDD4 antibody was used for immunoblotting analysis. Ponceau S staining was used as loading control

    Techniques Used: Expressing, Stable Transfection, Immunoprecipitation, SDS Page, Mutagenesis, Protein Extraction, Staining

    Degradation and poly-ubiquitination of Ezh1β are mainly contributed by NEDD4. a CHX chasing assay was performed in scramble/Ezh1β-FH and NEDD4 KD/Ezh1β-FH cell lines stressed with H 2 O 2 . scramble/Ezh1β-FH and NEDD4 KD/Ezh1β-FH cell lines were treated with 100 μM H 2 O 2 for 24 h. During last hour of H 2 O 2 treatment, 100 μg/ml cycloheximide (CHX) was added at indicated time points. Total proteins were extracted and immunoblot analysis was performed using anti-NEDD4 and anti-HA, anti-actin was used as loading control. b Quantification of remaining Ezh1β-FH protein percentage in a . Relative Ezh1β-FH was quantified in comparison remaining Ezh1β-FH with initial total protein amount at indicated CHX treatment time points. Data were expressed as mean ± SD from three biological replicates. ImageJ software was used to determine protein abundance. Values above each bar indicate Student’s t -test p value. c Poly-ubiquitination profile of Ezh1β under scramble and NEDD4 knock-down stable cell line. Scramble/1β-FH and NEDD4 KD/1β-FH indicate stable cell lines: scramble or NEDD4 KD constitutively expressing 1β-FH, respectively. Protein extracts were immunoprecipitated with FLAG and HA agarose beads and purified and ubiquitinated substrates were detected using anti-HA and anti-ubiquitin, respectively. Both scramble/1β-FH and NEDD4 KD/1β-FH stable cell lines were treated without or with H 2 O 2 were indicated as MT and H 2 O 2 . 10 μM MG-132 was treated for 4 h before protein extraction. Ponceau S staining was used as loading control
    Figure Legend Snippet: Degradation and poly-ubiquitination of Ezh1β are mainly contributed by NEDD4. a CHX chasing assay was performed in scramble/Ezh1β-FH and NEDD4 KD/Ezh1β-FH cell lines stressed with H 2 O 2 . scramble/Ezh1β-FH and NEDD4 KD/Ezh1β-FH cell lines were treated with 100 μM H 2 O 2 for 24 h. During last hour of H 2 O 2 treatment, 100 μg/ml cycloheximide (CHX) was added at indicated time points. Total proteins were extracted and immunoblot analysis was performed using anti-NEDD4 and anti-HA, anti-actin was used as loading control. b Quantification of remaining Ezh1β-FH protein percentage in a . Relative Ezh1β-FH was quantified in comparison remaining Ezh1β-FH with initial total protein amount at indicated CHX treatment time points. Data were expressed as mean ± SD from three biological replicates. ImageJ software was used to determine protein abundance. Values above each bar indicate Student’s t -test p value. c Poly-ubiquitination profile of Ezh1β under scramble and NEDD4 knock-down stable cell line. Scramble/1β-FH and NEDD4 KD/1β-FH indicate stable cell lines: scramble or NEDD4 KD constitutively expressing 1β-FH, respectively. Protein extracts were immunoprecipitated with FLAG and HA agarose beads and purified and ubiquitinated substrates were detected using anti-HA and anti-ubiquitin, respectively. Both scramble/1β-FH and NEDD4 KD/1β-FH stable cell lines were treated without or with H 2 O 2 were indicated as MT and H 2 O 2 . 10 μM MG-132 was treated for 4 h before protein extraction. Ponceau S staining was used as loading control

    Techniques Used: Software, Stable Transfection, Expressing, Immunoprecipitation, Purification, Protein Extraction, Staining

    34) Product Images from "RCAN1 Inhibits BACE2 Turnover by Attenuating Proteasome-Mediated BACE2 Degradation"

    Article Title: RCAN1 Inhibits BACE2 Turnover by Attenuating Proteasome-Mediated BACE2 Degradation

    Journal: BioMed Research International

    doi: 10.1155/2020/1920789

    RCAN1 attenuates proteasome-mediated BACE2 degradation. (a) HEK293 cells and (b) HRNLM cells were transfected with pBACE2-mycHis. 48 hours after transfection, cells were treated with 0, 10, 15, or 20 μ M proteasomal inhibitor MG-132 for 12 h. Cell lysates were resolved by 10% SDS-PAGE. 9E10 antibody was used to detect myc-tagged BACE2 protein. β -Actin served as an internal control. (c) Quantification of BACE2 levels. Values represent mean ± SEM; n ≥ 3, ∗ P
    Figure Legend Snippet: RCAN1 attenuates proteasome-mediated BACE2 degradation. (a) HEK293 cells and (b) HRNLM cells were transfected with pBACE2-mycHis. 48 hours after transfection, cells were treated with 0, 10, 15, or 20 μ M proteasomal inhibitor MG-132 for 12 h. Cell lysates were resolved by 10% SDS-PAGE. 9E10 antibody was used to detect myc-tagged BACE2 protein. β -Actin served as an internal control. (c) Quantification of BACE2 levels. Values represent mean ± SEM; n ≥ 3, ∗ P

    Techniques Used: Transfection, SDS Page

    35) Product Images from "Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells"

    Article Title: Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells

    Journal: Oncology Research

    doi: 10.3727/096504020X15929939001042

    MG-132 induces cell death in NSCLC cells. (A) H1299 and H441 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 1, and 5 μM) for 48 h. (B) A549 and H460 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 5, and 20 μM) for 48 h. Shown are representative images from three independent experiments. Scale bar: 400 μm. (C, D) Cell viability of H1299 and H441 cells were evaluated by cell counts. (E, F) Cell viability of A549 and H460 cells were evaluated by cell counts. Results are expressed as percent of cell viability normalized to control cells. The bar graphs represent mean with SD from three independent experiments. * p
    Figure Legend Snippet: MG-132 induces cell death in NSCLC cells. (A) H1299 and H441 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 1, and 5 μM) for 48 h. (B) A549 and H460 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 5, and 20 μM) for 48 h. Shown are representative images from three independent experiments. Scale bar: 400 μm. (C, D) Cell viability of H1299 and H441 cells were evaluated by cell counts. (E, F) Cell viability of A549 and H460 cells were evaluated by cell counts. Results are expressed as percent of cell viability normalized to control cells. The bar graphs represent mean with SD from three independent experiments. * p

    Techniques Used: Microscopy

    Proteasome inhibitors reduce c-Met expression in NSCLC cells. (A–D) H1299, H441, A549, and H460 cells were treated with increasing doses of bortezomib (0, 10, 50, and 100 nM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (E–H) H1299 and H441 cells were treated with increasing doses of MG-132 (0, 0.5, 1, 2, and 5 μM) for 48 h, while A549 and H460 cells were treated with higher doses of MG-132 (0, 5, 10, and 20 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (I–L) H1299, H441, A549, and H460 cells were treated with increasing doses of ONX-0914 (0, 1, 2, 5, and 10 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Shown are representative blots from three independent experiments. Intensities of blots were quantified by ImageJ software. Expression of c-Met was normalized to β-actin. The results are shown as mean ± SD of three independent experiments. * p
    Figure Legend Snippet: Proteasome inhibitors reduce c-Met expression in NSCLC cells. (A–D) H1299, H441, A549, and H460 cells were treated with increasing doses of bortezomib (0, 10, 50, and 100 nM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (E–H) H1299 and H441 cells were treated with increasing doses of MG-132 (0, 0.5, 1, 2, and 5 μM) for 48 h, while A549 and H460 cells were treated with higher doses of MG-132 (0, 5, 10, and 20 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (I–L) H1299, H441, A549, and H460 cells were treated with increasing doses of ONX-0914 (0, 1, 2, 5, and 10 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Shown are representative blots from three independent experiments. Intensities of blots were quantified by ImageJ software. Expression of c-Met was normalized to β-actin. The results are shown as mean ± SD of three independent experiments. * p

    Techniques Used: Expressing, Software

    Proteasome inhibitors increase PARP and caspase 3 cleavage and change p53 expression. (A) H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (B) H460 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (C) A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. Shown are representative blots from three independent experiments.
    Figure Legend Snippet: Proteasome inhibitors increase PARP and caspase 3 cleavage and change p53 expression. (A) H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (B) H460 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (C) A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. Shown are representative blots from three independent experiments.

    Techniques Used: Expressing

    The proteasome inhibitors have different effects on c-Met mRNA levels in NSCLC cells. (A–D) NSCLC cells were treated with 100 nM bortezomib for 48 h. c-Met mRNA levels were evaluated by real-time PCR. (E) c-Met mRNA levels in H1299 cells were evaluated after 5 μM MG-132 treatment for 48 h. (F) c-Met mRNA levels in H441 cells were evaluated after 2 μM MG-132 treatment for 48 h. (G) c-Met mRNA levels in H1299 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. (H) c-Met mRNA levels in H441 cells were evaluated after 5 μM ONX 0914 treatment for 48 h. (I) c-Met mRNA levels in H460 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. * p
    Figure Legend Snippet: The proteasome inhibitors have different effects on c-Met mRNA levels in NSCLC cells. (A–D) NSCLC cells were treated with 100 nM bortezomib for 48 h. c-Met mRNA levels were evaluated by real-time PCR. (E) c-Met mRNA levels in H1299 cells were evaluated after 5 μM MG-132 treatment for 48 h. (F) c-Met mRNA levels in H441 cells were evaluated after 2 μM MG-132 treatment for 48 h. (G) c-Met mRNA levels in H1299 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. (H) c-Met mRNA levels in H441 cells were evaluated after 5 μM ONX 0914 treatment for 48 h. (I) c-Met mRNA levels in H460 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. * p

    Techniques Used: Real-time Polymerase Chain Reaction

    Effect of proteasome inhibitors on CHX-mediated c-Met degradation. (A) H441 cells were treated with 20 μg/ml of CHX with or without MG132 (5 μM), bortezomib (100 nM), or ONX 0914 (10 μM). Cells were harvested at each time point over 0–8 h for immune blotting. Data represent one of three independent experiments with similar results. (B) Quantitative analysis of the immunoblots shown in (A) using ImageJ software. The results are shown as mean ± SD of three independent experiments. (C) A549 and H441 cells were transfected with c-Met-V5 plasmids for 1 day, and then were treated with proteasome inhibitors for an additional 48 h. H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM). A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM). Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Cell lysates were analyzed by immunoblotting with antibodies against V5 and β-actin.
    Figure Legend Snippet: Effect of proteasome inhibitors on CHX-mediated c-Met degradation. (A) H441 cells were treated with 20 μg/ml of CHX with or without MG132 (5 μM), bortezomib (100 nM), or ONX 0914 (10 μM). Cells were harvested at each time point over 0–8 h for immune blotting. Data represent one of three independent experiments with similar results. (B) Quantitative analysis of the immunoblots shown in (A) using ImageJ software. The results are shown as mean ± SD of three independent experiments. (C) A549 and H441 cells were transfected with c-Met-V5 plasmids for 1 day, and then were treated with proteasome inhibitors for an additional 48 h. H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM). A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM). Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Cell lysates were analyzed by immunoblotting with antibodies against V5 and β-actin.

    Techniques Used: Western Blot, Software, Transfection

    36) Product Images from "Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells"

    Article Title: Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells

    Journal: Oncology Research

    doi: 10.3727/096504020X15929939001042

    MG-132 induces cell death in NSCLC cells. (A) H1299 and H441 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 1, and 5 μM) for 48 h. (B) A549 and H460 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 5, and 20 μM) for 48 h. Shown are representative images from three independent experiments. Scale bar: 400 μm. (C, D) Cell viability of H1299 and H441 cells were evaluated by cell counts. (E, F) Cell viability of A549 and H460 cells were evaluated by cell counts. Results are expressed as percent of cell viability normalized to control cells. The bar graphs represent mean with SD from three independent experiments. * p
    Figure Legend Snippet: MG-132 induces cell death in NSCLC cells. (A) H1299 and H441 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 1, and 5 μM) for 48 h. (B) A549 and H460 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 5, and 20 μM) for 48 h. Shown are representative images from three independent experiments. Scale bar: 400 μm. (C, D) Cell viability of H1299 and H441 cells were evaluated by cell counts. (E, F) Cell viability of A549 and H460 cells were evaluated by cell counts. Results are expressed as percent of cell viability normalized to control cells. The bar graphs represent mean with SD from three independent experiments. * p

    Techniques Used: Microscopy

    Proteasome inhibitors reduce c-Met expression in NSCLC cells. (A–D) H1299, H441, A549, and H460 cells were treated with increasing doses of bortezomib (0, 10, 50, and 100 nM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (E–H) H1299 and H441 cells were treated with increasing doses of MG-132 (0, 0.5, 1, 2, and 5 μM) for 48 h, while A549 and H460 cells were treated with higher doses of MG-132 (0, 5, 10, and 20 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (I–L) H1299, H441, A549, and H460 cells were treated with increasing doses of ONX-0914 (0, 1, 2, 5, and 10 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Shown are representative blots from three independent experiments. Intensities of blots were quantified by ImageJ software. Expression of c-Met was normalized to β-actin. The results are shown as mean ± SD of three independent experiments. * p
    Figure Legend Snippet: Proteasome inhibitors reduce c-Met expression in NSCLC cells. (A–D) H1299, H441, A549, and H460 cells were treated with increasing doses of bortezomib (0, 10, 50, and 100 nM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (E–H) H1299 and H441 cells were treated with increasing doses of MG-132 (0, 0.5, 1, 2, and 5 μM) for 48 h, while A549 and H460 cells were treated with higher doses of MG-132 (0, 5, 10, and 20 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (I–L) H1299, H441, A549, and H460 cells were treated with increasing doses of ONX-0914 (0, 1, 2, 5, and 10 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Shown are representative blots from three independent experiments. Intensities of blots were quantified by ImageJ software. Expression of c-Met was normalized to β-actin. The results are shown as mean ± SD of three independent experiments. * p

    Techniques Used: Expressing, Software

    Proteasome inhibitors increase PARP and caspase 3 cleavage and change p53 expression. (A) H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (B) H460 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (C) A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. Shown are representative blots from three independent experiments.
    Figure Legend Snippet: Proteasome inhibitors increase PARP and caspase 3 cleavage and change p53 expression. (A) H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (B) H460 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (C) A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. Shown are representative blots from three independent experiments.

    Techniques Used: Expressing

    The proteasome inhibitors have different effects on c-Met mRNA levels in NSCLC cells. (A–D) NSCLC cells were treated with 100 nM bortezomib for 48 h. c-Met mRNA levels were evaluated by real-time PCR. (E) c-Met mRNA levels in H1299 cells were evaluated after 5 μM MG-132 treatment for 48 h. (F) c-Met mRNA levels in H441 cells were evaluated after 2 μM MG-132 treatment for 48 h. (G) c-Met mRNA levels in H1299 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. (H) c-Met mRNA levels in H441 cells were evaluated after 5 μM ONX 0914 treatment for 48 h. (I) c-Met mRNA levels in H460 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. * p
    Figure Legend Snippet: The proteasome inhibitors have different effects on c-Met mRNA levels in NSCLC cells. (A–D) NSCLC cells were treated with 100 nM bortezomib for 48 h. c-Met mRNA levels were evaluated by real-time PCR. (E) c-Met mRNA levels in H1299 cells were evaluated after 5 μM MG-132 treatment for 48 h. (F) c-Met mRNA levels in H441 cells were evaluated after 2 μM MG-132 treatment for 48 h. (G) c-Met mRNA levels in H1299 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. (H) c-Met mRNA levels in H441 cells were evaluated after 5 μM ONX 0914 treatment for 48 h. (I) c-Met mRNA levels in H460 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. * p

    Techniques Used: Real-time Polymerase Chain Reaction

    Effect of proteasome inhibitors on CHX-mediated c-Met degradation. (A) H441 cells were treated with 20 μg/ml of CHX with or without MG132 (5 μM), bortezomib (100 nM), or ONX 0914 (10 μM). Cells were harvested at each time point over 0–8 h for immune blotting. Data represent one of three independent experiments with similar results. (B) Quantitative analysis of the immunoblots shown in (A) using ImageJ software. The results are shown as mean ± SD of three independent experiments. (C) A549 and H441 cells were transfected with c-Met-V5 plasmids for 1 day, and then were treated with proteasome inhibitors for an additional 48 h. H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM). A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM). Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Cell lysates were analyzed by immunoblotting with antibodies against V5 and β-actin.
    Figure Legend Snippet: Effect of proteasome inhibitors on CHX-mediated c-Met degradation. (A) H441 cells were treated with 20 μg/ml of CHX with or without MG132 (5 μM), bortezomib (100 nM), or ONX 0914 (10 μM). Cells were harvested at each time point over 0–8 h for immune blotting. Data represent one of three independent experiments with similar results. (B) Quantitative analysis of the immunoblots shown in (A) using ImageJ software. The results are shown as mean ± SD of three independent experiments. (C) A549 and H441 cells were transfected with c-Met-V5 plasmids for 1 day, and then were treated with proteasome inhibitors for an additional 48 h. H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM). A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM). Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Cell lysates were analyzed by immunoblotting with antibodies against V5 and β-actin.

    Techniques Used: Western Blot, Software, Transfection

    37) Product Images from "Material aging causes centrosome weakening and disassembly during mitotic exit"

    Article Title: Material aging causes centrosome weakening and disassembly during mitotic exit

    Journal: bioRxiv

    doi: 10.1101/866434

    Acute inhibition of PLK-1 and SPD-2 induces premature weakening and disassembly of the PCM scaffold. A. PCM was subjected to high-flow FLUCS during metaphase in wild-type embryos or permeabilized embryos treated with 10 μM BI-2536 (inhibitor of Polo Kinases). Permeabilized embryos behaved as wild-type embryos during the first cell division (see methods; Carvalho 2011). B. PCM deformation rates in metaphase using low, medium, and high flow in wild-type and BI-2536-treated embryos. Wild-type data are from experiments in Figure Individual data points are plotted showing mean +/− 95% CI; n = 6-7 (wild-type) and n = 5-7 (BI-2536-treated). P-values were calculated using a Mann-Whitney test. C. PCM fracture probabilities from experiments in (B). D. Permeabilized embryos were arrested in metaphase using 10 μM MG-132, then treated with 0.1% ethanol (no drug) or 10 μM BI-2536. Data are plotted as normalized lines representing mean +/− 95% CI; n = 8 (no drug) and n = 10 (BI-2536-treated). E. Embryos expressing GFP::SPD-5 and a temperature-sensitive version of SPD-2 ( spd-2(or188ts) ) were allowed to assemble centrosomes at the permissive temperature (16°C), upshifted to the non-permissive temperature (25°C) for 1 min during prometaphase, then subjected to high-flow FLUCS during metaphase. F. For each experiment in spd-2(or188ts) embryos, one centrosome was subjected to FLUCS and the other left alone (control). Integrated fluorescent intensities of the SPD-5 signal were tracked over time, then normalized to the starting value. Each curve represents a single experiment. G. PCM fracture probabilities using low, medium, and high flow. Wild-type data are reproduced from Figure 1 . n = 7,7,8 (wild-type) and 5,6,8 ( spd-2(or188ts) ). H. Embryos were upshifted from 16°C to 23°C during metaphase, then imaged during anaphase. Data show integrated fluorescence densities of PCM-localized signal, plotted as normalized lines representing mean +/− 95% CI; n = 24 centrosomes in both wild-type and spd-2(or188ts) embryos.
    Figure Legend Snippet: Acute inhibition of PLK-1 and SPD-2 induces premature weakening and disassembly of the PCM scaffold. A. PCM was subjected to high-flow FLUCS during metaphase in wild-type embryos or permeabilized embryos treated with 10 μM BI-2536 (inhibitor of Polo Kinases). Permeabilized embryos behaved as wild-type embryos during the first cell division (see methods; Carvalho 2011). B. PCM deformation rates in metaphase using low, medium, and high flow in wild-type and BI-2536-treated embryos. Wild-type data are from experiments in Figure Individual data points are plotted showing mean +/− 95% CI; n = 6-7 (wild-type) and n = 5-7 (BI-2536-treated). P-values were calculated using a Mann-Whitney test. C. PCM fracture probabilities from experiments in (B). D. Permeabilized embryos were arrested in metaphase using 10 μM MG-132, then treated with 0.1% ethanol (no drug) or 10 μM BI-2536. Data are plotted as normalized lines representing mean +/− 95% CI; n = 8 (no drug) and n = 10 (BI-2536-treated). E. Embryos expressing GFP::SPD-5 and a temperature-sensitive version of SPD-2 ( spd-2(or188ts) ) were allowed to assemble centrosomes at the permissive temperature (16°C), upshifted to the non-permissive temperature (25°C) for 1 min during prometaphase, then subjected to high-flow FLUCS during metaphase. F. For each experiment in spd-2(or188ts) embryos, one centrosome was subjected to FLUCS and the other left alone (control). Integrated fluorescent intensities of the SPD-5 signal were tracked over time, then normalized to the starting value. Each curve represents a single experiment. G. PCM fracture probabilities using low, medium, and high flow. Wild-type data are reproduced from Figure 1 . n = 7,7,8 (wild-type) and 5,6,8 ( spd-2(or188ts) ). H. Embryos were upshifted from 16°C to 23°C during metaphase, then imaged during anaphase. Data show integrated fluorescence densities of PCM-localized signal, plotted as normalized lines representing mean +/− 95% CI; n = 24 centrosomes in both wild-type and spd-2(or188ts) embryos.

    Techniques Used: Inhibition, MANN-WHITNEY, Expressing, Fluorescence

    38) Product Images from "Cytotoxic Activity of Aplykurodin A Isolated From Aplysia kurodai against AXIN1-Mutated Hepatocellular Carcinoma Cells by Promoting Oncogenic β-Catenin Degradation"

    Article Title: Cytotoxic Activity of Aplykurodin A Isolated From Aplysia kurodai against AXIN1-Mutated Hepatocellular Carcinoma Cells by Promoting Oncogenic β-Catenin Degradation

    Journal: Marine Drugs

    doi: 10.3390/md18040210

    Aplykurodin A promotes proteasomal β-catenin degradation. ( A ) After treatment of HEK293-FL cells with either DMSO or aplykurodin A (20 and 40 μM) in the presence of Wnt3a-CM for 15 h, cytosolic proteins were analyzed by Western blotting with anti-β-catenin antibody. ( B ) After treatment of HEK293-FL cells with either DMSO or aplykurodin A (20 and 40 μM) in the presence of Wnt3a-CM for 15 h, semi-quantitative RT-PCRs for β-catenin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were carried out with total RNA. ( C ) HEK293-FL reporter cells were treated with either DMSO or aplykurodin A (20 μM) and then exposed to MG-132 (10 μM) for 8 h. Cytosolic proteins were analyzed by Western blotting with anti-β-catenin antibody. ( A , C ) the blots were re-probed with anti-actin antibody. The results are representative of three independent experiments.
    Figure Legend Snippet: Aplykurodin A promotes proteasomal β-catenin degradation. ( A ) After treatment of HEK293-FL cells with either DMSO or aplykurodin A (20 and 40 μM) in the presence of Wnt3a-CM for 15 h, cytosolic proteins were analyzed by Western blotting with anti-β-catenin antibody. ( B ) After treatment of HEK293-FL cells with either DMSO or aplykurodin A (20 and 40 μM) in the presence of Wnt3a-CM for 15 h, semi-quantitative RT-PCRs for β-catenin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were carried out with total RNA. ( C ) HEK293-FL reporter cells were treated with either DMSO or aplykurodin A (20 μM) and then exposed to MG-132 (10 μM) for 8 h. Cytosolic proteins were analyzed by Western blotting with anti-β-catenin antibody. ( A , C ) the blots were re-probed with anti-actin antibody. The results are representative of three independent experiments.

    Techniques Used: Western Blot

    39) Product Images from "mTORC2 mediate FLCN-induced HIF2α nuclear import and proliferation of clear cell renal cell carcinoma"

    Article Title: mTORC2 mediate FLCN-induced HIF2α nuclear import and proliferation of clear cell renal cell carcinoma

    Journal: bioRxiv

    doi: 10.1101/2020.01.13.905521

    FLCN knockdown restricts HIF2α degradation of ccRCC cells. (A) Western blot showing FLCN co-IP HIF2α or HIF2α co-IP FLCN in 786-O, ACHN and HK-2 cells. IgG as a control. (B,C) 786-O and ACHN cells transfected with control siRNA or siFLCN overnight. In addition to its protein synthesis blocked with cycloheximide (CHX, 10 μg/mL) for the indicated times. Then, the cells were lysed and HIF2α level was determined by Western blotting (B). HIF2α bands were quantified and normalized against β-actin (C). (D,E) 786-O and ACHN cells in addition to its Ubiquitination inhibitors with DMSO or MG-132 (5μg/mL), the cells were then incubated KU0063794 (10 ng/mL) for the indicated times. Then, the cells were lysed and HIF2α level was determined by Western blotting (D). HIF2α bands were quantified and normalized against β-actin (E). The data was from three independently repeated experiments. *: P
    Figure Legend Snippet: FLCN knockdown restricts HIF2α degradation of ccRCC cells. (A) Western blot showing FLCN co-IP HIF2α or HIF2α co-IP FLCN in 786-O, ACHN and HK-2 cells. IgG as a control. (B,C) 786-O and ACHN cells transfected with control siRNA or siFLCN overnight. In addition to its protein synthesis blocked with cycloheximide (CHX, 10 μg/mL) for the indicated times. Then, the cells were lysed and HIF2α level was determined by Western blotting (B). HIF2α bands were quantified and normalized against β-actin (C). (D,E) 786-O and ACHN cells in addition to its Ubiquitination inhibitors with DMSO or MG-132 (5μg/mL), the cells were then incubated KU0063794 (10 ng/mL) for the indicated times. Then, the cells were lysed and HIF2α level was determined by Western blotting (D). HIF2α bands were quantified and normalized against β-actin (E). The data was from three independently repeated experiments. *: P

    Techniques Used: Western Blot, Co-Immunoprecipitation Assay, Transfection, Incubation

    40) Product Images from "Downregulation of TRAF2 Mediates NIK-Induced Pancreatic Cancer Cell Proliferation and Tumorigenicity"

    Article Title: Downregulation of TRAF2 Mediates NIK-Induced Pancreatic Cancer Cell Proliferation and Tumorigenicity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0053676

    TRAF2 expression is downregulated in PDAC cell lines. A: Cell lysates of indicated cells were normalized to 0.5 mg/ml and then 20 µg were subjected to SDS-PAGE. Samples were transferred to nitrocellulose and analyzed by Western blot for expression of TRAF2 (anti-TRAF2), TRAF3 (anti-TRAF3), cIAP1 (anti-cIAP1), cIAP2 (anti-cIAP2) or β-actin (anti-β-actin; loading control). TRAF2 overexpressed in Hek293 cells served as an additional molecular weight control. B: Samples of mRNA of indicated cell lines were subjected to quantitative RT-PCR directed against TRAF2. Samples were normalized to GAPDH. C: Panc1 cells (5×10 5 cells, 6 cm dishes) were co-transfected with TRAF2 and vector control or HA-ubiquitin. After 24 hours cells were lysed, TRAF2 immunoprecipitated (anti-TRAF2) and analyzed by immunoblotting for ubiquitination of TRAF2 (anti-HA). Blots were re-probed for TRAF2 (anti-TRAF2). D: Indicated cell lines were treated with MG-132 (20 µM) for 0, 4, 8, 16 or 24 hours. Cells were lysed and analyzed for expression of endogenous TRAF2 (anti-TRAF2) or β-actin (anti-β-actin; loading control. TRAF2 overexpressed in Hek293 cells served as positive control. E: Panc1 cells (5×10 5 cells, 6 cm dishes) were transfected with vector control or TRAF2 as indicated. After 24 hours cell lysates were analyzed by Western blotting for expression of NIK (anti-NIK), overexpressed TRAF2 (anti-TRAF2) or β-actin (anti-β-actin) as loading control.
    Figure Legend Snippet: TRAF2 expression is downregulated in PDAC cell lines. A: Cell lysates of indicated cells were normalized to 0.5 mg/ml and then 20 µg were subjected to SDS-PAGE. Samples were transferred to nitrocellulose and analyzed by Western blot for expression of TRAF2 (anti-TRAF2), TRAF3 (anti-TRAF3), cIAP1 (anti-cIAP1), cIAP2 (anti-cIAP2) or β-actin (anti-β-actin; loading control). TRAF2 overexpressed in Hek293 cells served as an additional molecular weight control. B: Samples of mRNA of indicated cell lines were subjected to quantitative RT-PCR directed against TRAF2. Samples were normalized to GAPDH. C: Panc1 cells (5×10 5 cells, 6 cm dishes) were co-transfected with TRAF2 and vector control or HA-ubiquitin. After 24 hours cells were lysed, TRAF2 immunoprecipitated (anti-TRAF2) and analyzed by immunoblotting for ubiquitination of TRAF2 (anti-HA). Blots were re-probed for TRAF2 (anti-TRAF2). D: Indicated cell lines were treated with MG-132 (20 µM) for 0, 4, 8, 16 or 24 hours. Cells were lysed and analyzed for expression of endogenous TRAF2 (anti-TRAF2) or β-actin (anti-β-actin; loading control. TRAF2 overexpressed in Hek293 cells served as positive control. E: Panc1 cells (5×10 5 cells, 6 cm dishes) were transfected with vector control or TRAF2 as indicated. After 24 hours cell lysates were analyzed by Western blotting for expression of NIK (anti-NIK), overexpressed TRAF2 (anti-TRAF2) or β-actin (anti-β-actin) as loading control.

    Techniques Used: Expressing, SDS Page, Western Blot, Molecular Weight, Quantitative RT-PCR, Transfection, Plasmid Preparation, Immunoprecipitation, Positive Control

    Related Articles

    Protease Inhibitor:

    Article Title: FERONIA Confers Resistance to Photooxidative Stress in Arabidopsis
    Article Snippet: .. Col-0 and FERpro :FER-MYC seedlings were ground in liquid nitrogen and 2 volumes of IP buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10% (v/v) glycerol, 5 mM EDTA, 1% (v/v) Triton X-100, 1% (v/v) NP-40, 1 × protease inhibitor cocktail (Sigma-Aldrich), and 50 μM MG132 (Sigma-Aldrich) were added for total protein extraction. ..

    Protein Extraction:

    Article Title: FERONIA Confers Resistance to Photooxidative Stress in Arabidopsis
    Article Snippet: .. Col-0 and FERpro :FER-MYC seedlings were ground in liquid nitrogen and 2 volumes of IP buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10% (v/v) glycerol, 5 mM EDTA, 1% (v/v) Triton X-100, 1% (v/v) NP-40, 1 × protease inhibitor cocktail (Sigma-Aldrich), and 50 μM MG132 (Sigma-Aldrich) were added for total protein extraction. ..

    other:

    Article Title: Cullin 1 (CUL1) Promotes Primary Ciliogenesis through the Induction of Ubiquitin-Proteasome-Dependent Dvl2 Degradation
    Article Snippet: To inhibit proteasomal degradation pathway, cells were treated with 10 μM of MG132 (Calbiochem, San Diego, CA, USA), 100 μM of ALLN (Sigma, St. Louis, MO, USA), or 10 μM of lactacystin (Sigma, St. Louis, MO, USA) for 3 h. To inhibit autophagy, cells were treated with 0.2 μM of bafilomycin A1 (Sigma, St. Louis, MO, USA) or 100 μM of chloroquine (Sigma, St. Louis, MO, USA) for 3 h. To inhibit NEDD8-activating Enzyme (NAE), cells were treated with 0.5 or 1 μM of MLN4924 (Calbiochem, San Diego, CA, USA).

    Activity Assay:

    Article Title: A model of the aged lung epithelium in idiopathic pulmonary fibrosis
    Article Snippet: .. Proteasome activity was determined as the difference between the total activity in the presence or absence of 20 μM MG132 (Millipore-Sigma). ..

    Transfection:

    Article Title: Prostate cancer-associated SPOP mutations lead to genomic instability through disruption of the SPOP–HIPK2 axis
    Article Snippet: .. Thirty-six hours after transfection, cells were treated with 30 μM MG132 for 6 h and then lysed in 1% SDS buffer (Tris [pH 7.5], 0.5 mM EDTA, 1 mM DTT) and boiled for 10 min. For immunoprecipitation, the cell lysates were diluted 10-fold in Tris–HCl buffer and incubated with anti-FLAG M2 agarose beads (Sigma) for 4 h at 4°C. ..

    Article Title: MiR-195 inhibits the ubiquitination and degradation of YY1 by Smurf2, and induces EMT and cell permeability of retinal pigment epithelial cells
    Article Snippet: .. ARPE-19 cells were transfected with 20 nM of oligonucleotides or 600 ng vectors using Lipofectamine 3000 (Thermo Fisher) for 24 h. MG132 was purchased from Sigma. ..

    Immunoprecipitation:

    Article Title: Prostate cancer-associated SPOP mutations lead to genomic instability through disruption of the SPOP–HIPK2 axis
    Article Snippet: .. Thirty-six hours after transfection, cells were treated with 30 μM MG132 for 6 h and then lysed in 1% SDS buffer (Tris [pH 7.5], 0.5 mM EDTA, 1 mM DTT) and boiled for 10 min. For immunoprecipitation, the cell lysates were diluted 10-fold in Tris–HCl buffer and incubated with anti-FLAG M2 agarose beads (Sigma) for 4 h at 4°C. ..

    Incubation:

    Article Title: Prostate cancer-associated SPOP mutations lead to genomic instability through disruption of the SPOP–HIPK2 axis
    Article Snippet: .. Thirty-six hours after transfection, cells were treated with 30 μM MG132 for 6 h and then lysed in 1% SDS buffer (Tris [pH 7.5], 0.5 mM EDTA, 1 mM DTT) and boiled for 10 min. For immunoprecipitation, the cell lysates were diluted 10-fold in Tris–HCl buffer and incubated with anti-FLAG M2 agarose beads (Sigma) for 4 h at 4°C. ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    Millipore mg 132
    <t>MG-132</t> induces cell death in NSCLC cells. (A) H1299 and H441 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 1, and 5 μM) for 48 h. (B) A549 and H460 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 5, and 20 μM) for 48 h. Shown are representative images from three independent experiments. Scale bar: 400 μm. (C, D) Cell viability of H1299 and H441 cells were evaluated by cell counts. (E, F) Cell viability of A549 and H460 cells were evaluated by cell counts. Results are expressed as percent of cell viability normalized to control cells. The bar graphs represent mean with SD from three independent experiments. * p
    Mg 132, 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/mg 132/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mg 132 - by Bioz Stars, 2021-09
    97/100 stars
      Buy from Supplier

    99
    Millipore bay11 7082
    MPs released by LPS-treated THP-1 cells induce the expression of cell adhesion molecules by HUVECs. (A) HUVECs were incubated for 1 or 2 hours in serum-free conditions with MPs from untreated or LPS-treated THP-1 cells or 10 ng/mL TNF-α. ICAM-1 , VCAM-1 , and E-selectin mRNA expression was measured by real-time polymerase chain reaction. (B) HUVECs were incubated for 4 hours in serum-free conditions with MPs derived from LPS-treated or untreated THP-1 cells or 10 ng/mL TNF-α. Expression of VCAM-1, E-selectin, and ICAM-1 by HUVECs was detected by Western blot. (C) HUVECs were incubated for 4 hours in serum-free conditions with MPs from LPS-treated PBMCs or 10 ng/mL TNF-α. VCAM-1 was detected by Western blot. (D) HUVECs were treated with 5 or 10μM of the NF-κB pathway inhibitors MG-132, <t>Bay11–7082,</t> or vehicle control (dimethyl sulfoxide [DMSO]) and then stimulated with MPs from LPS-treated THP-1 cells. VCAM-1 was detected by Western blot. GAPDH served as a loading control. Results are representative of 3 independent experiments.
    Bay11 7082, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bay11 7082/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bay11 7082 - by Bioz Stars, 2021-09
    99/100 stars
      Buy from Supplier

    99
    Millipore mg132
    Cul7 degrades rEag1 via both proteasomal and lysosomal pathways. ( A ) Proteasome inhibition with 10 μM <t>MG132</t> (in DMSO). In the absence of MG132, rEag1 expression in HEK293T cells is characterized by two protein bands (a, b) of 110–120 kDa. Increasing MG132 treatment durations leads to enhanced bands a and b signals, as well as the presence of an additional band ( c ) with the lowest molecular weight. ( B ) Glycan structure modification with tunicamycin, Endo H, or PNGase F exerts distinct effects on the three rEag1 protein bands. ( C ) PNGase F and MG132 treatments result in the appearance of apparently identical rEag1 protein band c. ( D ) MG132 treatment reverses Cul7 effect on rEag1 protein band b. ( Left ) Representative immunoblots showing Cul7 effects in the absence or presence of MG132 treatment. Each experimental result is displayed in duplicates. ( Right ) Quantification of Cul7 effects on the three rEag1 protein bands in the absence or presence of MG132 treatment (see Suppl. Fig. S5A for detailed differentiation of rEag1 bands) (*p
    Mg132, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mg132/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mg132 - by Bioz Stars, 2021-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    MG-132 induces cell death in NSCLC cells. (A) H1299 and H441 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 1, and 5 μM) for 48 h. (B) A549 and H460 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 5, and 20 μM) for 48 h. Shown are representative images from three independent experiments. Scale bar: 400 μm. (C, D) Cell viability of H1299 and H441 cells were evaluated by cell counts. (E, F) Cell viability of A549 and H460 cells were evaluated by cell counts. Results are expressed as percent of cell viability normalized to control cells. The bar graphs represent mean with SD from three independent experiments. * p

    Journal: Oncology Research

    Article Title: Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells

    doi: 10.3727/096504020X15929939001042

    Figure Lengend Snippet: MG-132 induces cell death in NSCLC cells. (A) H1299 and H441 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 1, and 5 μM) for 48 h. (B) A549 and H460 cells were pictured under a phase-contrast microscope after treatment with MG-132 at different concentrations (0, 5, and 20 μM) for 48 h. Shown are representative images from three independent experiments. Scale bar: 400 μm. (C, D) Cell viability of H1299 and H441 cells were evaluated by cell counts. (E, F) Cell viability of A549 and H460 cells were evaluated by cell counts. Results are expressed as percent of cell viability normalized to control cells. The bar graphs represent mean with SD from three independent experiments. * p

    Article Snippet: MG-132 at a lower concentration (1 μM) diminished c-Met levels in H441 cells , while MG-132 decreased c-Met levels in H1299 cells at 5 μM concentration ( ).

    Techniques: Microscopy

    Proteasome inhibitors reduce c-Met expression in NSCLC cells. (A–D) H1299, H441, A549, and H460 cells were treated with increasing doses of bortezomib (0, 10, 50, and 100 nM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (E–H) H1299 and H441 cells were treated with increasing doses of MG-132 (0, 0.5, 1, 2, and 5 μM) for 48 h, while A549 and H460 cells were treated with higher doses of MG-132 (0, 5, 10, and 20 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (I–L) H1299, H441, A549, and H460 cells were treated with increasing doses of ONX-0914 (0, 1, 2, 5, and 10 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Shown are representative blots from three independent experiments. Intensities of blots were quantified by ImageJ software. Expression of c-Met was normalized to β-actin. The results are shown as mean ± SD of three independent experiments. * p

    Journal: Oncology Research

    Article Title: Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells

    doi: 10.3727/096504020X15929939001042

    Figure Lengend Snippet: Proteasome inhibitors reduce c-Met expression in NSCLC cells. (A–D) H1299, H441, A549, and H460 cells were treated with increasing doses of bortezomib (0, 10, 50, and 100 nM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (E–H) H1299 and H441 cells were treated with increasing doses of MG-132 (0, 0.5, 1, 2, and 5 μM) for 48 h, while A549 and H460 cells were treated with higher doses of MG-132 (0, 5, 10, and 20 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. (I–L) H1299, H441, A549, and H460 cells were treated with increasing doses of ONX-0914 (0, 1, 2, 5, and 10 μM) for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Shown are representative blots from three independent experiments. Intensities of blots were quantified by ImageJ software. Expression of c-Met was normalized to β-actin. The results are shown as mean ± SD of three independent experiments. * p

    Article Snippet: MG-132 at a lower concentration (1 μM) diminished c-Met levels in H441 cells , while MG-132 decreased c-Met levels in H1299 cells at 5 μM concentration ( ).

    Techniques: Expressing, Software

    Proteasome inhibitors increase PARP and caspase 3 cleavage and change p53 expression. (A) H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (B) H460 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (C) A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. Shown are representative blots from three independent experiments.

    Journal: Oncology Research

    Article Title: Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells

    doi: 10.3727/096504020X15929939001042

    Figure Lengend Snippet: Proteasome inhibitors increase PARP and caspase 3 cleavage and change p53 expression. (A) H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (B) H460 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. (C) A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM), respectively, for 48 h, then were challenged with HGF (50 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with antibodies against c-Met, p-c-Met, PARP, p53, cleaved caspase 3, and β-actin. Shown are representative blots from three independent experiments.

    Article Snippet: MG-132 at a lower concentration (1 μM) diminished c-Met levels in H441 cells , while MG-132 decreased c-Met levels in H1299 cells at 5 μM concentration ( ).

    Techniques: Expressing

    The proteasome inhibitors have different effects on c-Met mRNA levels in NSCLC cells. (A–D) NSCLC cells were treated with 100 nM bortezomib for 48 h. c-Met mRNA levels were evaluated by real-time PCR. (E) c-Met mRNA levels in H1299 cells were evaluated after 5 μM MG-132 treatment for 48 h. (F) c-Met mRNA levels in H441 cells were evaluated after 2 μM MG-132 treatment for 48 h. (G) c-Met mRNA levels in H1299 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. (H) c-Met mRNA levels in H441 cells were evaluated after 5 μM ONX 0914 treatment for 48 h. (I) c-Met mRNA levels in H460 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. * p

    Journal: Oncology Research

    Article Title: Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells

    doi: 10.3727/096504020X15929939001042

    Figure Lengend Snippet: The proteasome inhibitors have different effects on c-Met mRNA levels in NSCLC cells. (A–D) NSCLC cells were treated with 100 nM bortezomib for 48 h. c-Met mRNA levels were evaluated by real-time PCR. (E) c-Met mRNA levels in H1299 cells were evaluated after 5 μM MG-132 treatment for 48 h. (F) c-Met mRNA levels in H441 cells were evaluated after 2 μM MG-132 treatment for 48 h. (G) c-Met mRNA levels in H1299 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. (H) c-Met mRNA levels in H441 cells were evaluated after 5 μM ONX 0914 treatment for 48 h. (I) c-Met mRNA levels in H460 cells were evaluated after 10 μM ONX 0914 treatment for 48 h. * p

    Article Snippet: MG-132 at a lower concentration (1 μM) diminished c-Met levels in H441 cells , while MG-132 decreased c-Met levels in H1299 cells at 5 μM concentration ( ).

    Techniques: Real-time Polymerase Chain Reaction

    Effect of proteasome inhibitors on CHX-mediated c-Met degradation. (A) H441 cells were treated with 20 μg/ml of CHX with or without MG132 (5 μM), bortezomib (100 nM), or ONX 0914 (10 μM). Cells were harvested at each time point over 0–8 h for immune blotting. Data represent one of three independent experiments with similar results. (B) Quantitative analysis of the immunoblots shown in (A) using ImageJ software. The results are shown as mean ± SD of three independent experiments. (C) A549 and H441 cells were transfected with c-Met-V5 plasmids for 1 day, and then were treated with proteasome inhibitors for an additional 48 h. H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM). A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM). Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Cell lysates were analyzed by immunoblotting with antibodies against V5 and β-actin.

    Journal: Oncology Research

    Article Title: Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells

    doi: 10.3727/096504020X15929939001042

    Figure Lengend Snippet: Effect of proteasome inhibitors on CHX-mediated c-Met degradation. (A) H441 cells were treated with 20 μg/ml of CHX with or without MG132 (5 μM), bortezomib (100 nM), or ONX 0914 (10 μM). Cells were harvested at each time point over 0–8 h for immune blotting. Data represent one of three independent experiments with similar results. (B) Quantitative analysis of the immunoblots shown in (A) using ImageJ software. The results are shown as mean ± SD of three independent experiments. (C) A549 and H441 cells were transfected with c-Met-V5 plasmids for 1 day, and then were treated with proteasome inhibitors for an additional 48 h. H441 cells were treated with MG-132 (5 μM), bortezomib (100 nM), and ONX 0914 (10 μM). A549 cells were treated with MG-132 (20 μM), bortezomib (100 nM), and ONX 0914 (10 μM). Cell lysates were analyzed by immunoblotting with antibodies against c-Met and β-actin. Cell lysates were analyzed by immunoblotting with antibodies against V5 and β-actin.

    Article Snippet: MG-132 at a lower concentration (1 μM) diminished c-Met levels in H441 cells , while MG-132 decreased c-Met levels in H1299 cells at 5 μM concentration ( ).

    Techniques: Western Blot, Software, Transfection

    MPs released by LPS-treated THP-1 cells induce the expression of cell adhesion molecules by HUVECs. (A) HUVECs were incubated for 1 or 2 hours in serum-free conditions with MPs from untreated or LPS-treated THP-1 cells or 10 ng/mL TNF-α. ICAM-1 , VCAM-1 , and E-selectin mRNA expression was measured by real-time polymerase chain reaction. (B) HUVECs were incubated for 4 hours in serum-free conditions with MPs derived from LPS-treated or untreated THP-1 cells or 10 ng/mL TNF-α. Expression of VCAM-1, E-selectin, and ICAM-1 by HUVECs was detected by Western blot. (C) HUVECs were incubated for 4 hours in serum-free conditions with MPs from LPS-treated PBMCs or 10 ng/mL TNF-α. VCAM-1 was detected by Western blot. (D) HUVECs were treated with 5 or 10μM of the NF-κB pathway inhibitors MG-132, Bay11–7082, or vehicle control (dimethyl sulfoxide [DMSO]) and then stimulated with MPs from LPS-treated THP-1 cells. VCAM-1 was detected by Western blot. GAPDH served as a loading control. Results are representative of 3 independent experiments.

    Journal: Blood

    Article Title: Monocytic microparticles activate endothelial cells in an IL-1?-dependent manner

    doi: 10.1182/blood-2011-01-330878

    Figure Lengend Snippet: MPs released by LPS-treated THP-1 cells induce the expression of cell adhesion molecules by HUVECs. (A) HUVECs were incubated for 1 or 2 hours in serum-free conditions with MPs from untreated or LPS-treated THP-1 cells or 10 ng/mL TNF-α. ICAM-1 , VCAM-1 , and E-selectin mRNA expression was measured by real-time polymerase chain reaction. (B) HUVECs were incubated for 4 hours in serum-free conditions with MPs derived from LPS-treated or untreated THP-1 cells or 10 ng/mL TNF-α. Expression of VCAM-1, E-selectin, and ICAM-1 by HUVECs was detected by Western blot. (C) HUVECs were incubated for 4 hours in serum-free conditions with MPs from LPS-treated PBMCs or 10 ng/mL TNF-α. VCAM-1 was detected by Western blot. (D) HUVECs were treated with 5 or 10μM of the NF-κB pathway inhibitors MG-132, Bay11–7082, or vehicle control (dimethyl sulfoxide [DMSO]) and then stimulated with MPs from LPS-treated THP-1 cells. VCAM-1 was detected by Western blot. GAPDH served as a loading control. Results are representative of 3 independent experiments.

    Article Snippet: LPS ( Escherichia coli serotype O111:B4), MG-132, and Bay11–7082 were obtained from Sigma-Aldrich.

    Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Derivative Assay, Western Blot

    Cul7 degrades rEag1 via both proteasomal and lysosomal pathways. ( A ) Proteasome inhibition with 10 μM MG132 (in DMSO). In the absence of MG132, rEag1 expression in HEK293T cells is characterized by two protein bands (a, b) of 110–120 kDa. Increasing MG132 treatment durations leads to enhanced bands a and b signals, as well as the presence of an additional band ( c ) with the lowest molecular weight. ( B ) Glycan structure modification with tunicamycin, Endo H, or PNGase F exerts distinct effects on the three rEag1 protein bands. ( C ) PNGase F and MG132 treatments result in the appearance of apparently identical rEag1 protein band c. ( D ) MG132 treatment reverses Cul7 effect on rEag1 protein band b. ( Left ) Representative immunoblots showing Cul7 effects in the absence or presence of MG132 treatment. Each experimental result is displayed in duplicates. ( Right ) Quantification of Cul7 effects on the three rEag1 protein bands in the absence or presence of MG132 treatment (see Suppl. Fig. S5A for detailed differentiation of rEag1 bands) (*p

    Journal: Scientific Reports

    Article Title: Cullin 7 mediates proteasomal and lysosomal degradations of rat Eag1 potassium channels

    doi: 10.1038/srep40825

    Figure Lengend Snippet: Cul7 degrades rEag1 via both proteasomal and lysosomal pathways. ( A ) Proteasome inhibition with 10 μM MG132 (in DMSO). In the absence of MG132, rEag1 expression in HEK293T cells is characterized by two protein bands (a, b) of 110–120 kDa. Increasing MG132 treatment durations leads to enhanced bands a and b signals, as well as the presence of an additional band ( c ) with the lowest molecular weight. ( B ) Glycan structure modification with tunicamycin, Endo H, or PNGase F exerts distinct effects on the three rEag1 protein bands. ( C ) PNGase F and MG132 treatments result in the appearance of apparently identical rEag1 protein band c. ( D ) MG132 treatment reverses Cul7 effect on rEag1 protein band b. ( Left ) Representative immunoblots showing Cul7 effects in the absence or presence of MG132 treatment. Each experimental result is displayed in duplicates. ( Right ) Quantification of Cul7 effects on the three rEag1 protein bands in the absence or presence of MG132 treatment (see Suppl. Fig. S5A for detailed differentiation of rEag1 bands) (*p

    Article Snippet: Where indicated, cells were treated with ALLN (Sigma), brefeldin A (Sigma), chloroquine (Sigma), cycloheximide (Sigma), MG132 (Sigma), MLN4924 (Dr. Kuo-How Huang, National Taiwan University Hospital), or tunicamycin (Sigma).

    Techniques: Inhibition, Expressing, Molecular Weight, Modification, Western Blot

    Pharmacological suppression of proteasomal and lysosomal degradations alters subcellular localization of rEag1 in HEK293T cells. Representative confocal micrographs showing rEag1 immunofluorescence signals (green) in response to Cul7 co-expression ( A , B , C ), or 6-hour MG132 ( D , E , F ) or chloroquine ( G , H , I ) treatment. rEag1 and Myc-Cul7 were detected with anti-rEag1 and anti-Myc antibodies, respectively. Nuclei were counterstained with DAPI (blue). To verify plasma membrane localization, some cells were co-transfected with the DsRed-membrane expression vector (DsRed-Mem). ER and lysosomal localizations were detected by specific antibodies for the ER marker calnexin and the lysosome marker lamp1, respectively. Merge images are shown in the third column of each panel. Arrowheads indicate plasma membrane staining, whereas arrows denote intracellular staining. See Supplementary Methods and Supplementary Figure S2B for further quantitative analyses. Scale bar, 10 μm. Data shown here are representative of over 80 cells from at least 3 independent experiments.

    Journal: Scientific Reports

    Article Title: Cullin 7 mediates proteasomal and lysosomal degradations of rat Eag1 potassium channels

    doi: 10.1038/srep40825

    Figure Lengend Snippet: Pharmacological suppression of proteasomal and lysosomal degradations alters subcellular localization of rEag1 in HEK293T cells. Representative confocal micrographs showing rEag1 immunofluorescence signals (green) in response to Cul7 co-expression ( A , B , C ), or 6-hour MG132 ( D , E , F ) or chloroquine ( G , H , I ) treatment. rEag1 and Myc-Cul7 were detected with anti-rEag1 and anti-Myc antibodies, respectively. Nuclei were counterstained with DAPI (blue). To verify plasma membrane localization, some cells were co-transfected with the DsRed-membrane expression vector (DsRed-Mem). ER and lysosomal localizations were detected by specific antibodies for the ER marker calnexin and the lysosome marker lamp1, respectively. Merge images are shown in the third column of each panel. Arrowheads indicate plasma membrane staining, whereas arrows denote intracellular staining. See Supplementary Methods and Supplementary Figure S2B for further quantitative analyses. Scale bar, 10 μm. Data shown here are representative of over 80 cells from at least 3 independent experiments.

    Article Snippet: Where indicated, cells were treated with ALLN (Sigma), brefeldin A (Sigma), chloroquine (Sigma), cycloheximide (Sigma), MG132 (Sigma), MLN4924 (Dr. Kuo-How Huang, National Taiwan University Hospital), or tunicamycin (Sigma).

    Techniques: Immunofluorescence, Expressing, Transfection, Plasmid Preparation, Marker, Staining

    Ataxin-3 targets mutant SOD1 to aggresomes to with autophagic activity. A , 293 cells co-expressing RFP-tagged SOD1 G85R , EGFP-tagged ataxin-3 and Flag-tagged p62 were incubated in 10 μ m MG132 for 12 h. The cells were then fixed and stained with

    Journal: The Journal of Biological Chemistry

    Article Title: Ataxin-3 Regulates Aggresome Formation of Copper-Zinc Superoxide Dismutase (SOD1) by Editing K63-linked Polyubiquitin Chains *

    doi: 10.1074/jbc.M111.299990

    Figure Lengend Snippet: Ataxin-3 targets mutant SOD1 to aggresomes to with autophagic activity. A , 293 cells co-expressing RFP-tagged SOD1 G85R , EGFP-tagged ataxin-3 and Flag-tagged p62 were incubated in 10 μ m MG132 for 12 h. The cells were then fixed and stained with

    Article Snippet: MG132 was purchased from Calbiochem.

    Techniques: Mutagenesis, Activity Assay, Expressing, Incubation, Staining

    Colocalization of mutant SOD1 and ataxin-3 in aggresomes. A , 293 cells were transfected with EGFP or SOD1 G85R -EGFP and treated with 10 μ m MG132 for 12 h. The supernatants of the cell lysates were immunoprecipitated with rabbit polyclonal antibodies

    Journal: The Journal of Biological Chemistry

    Article Title: Ataxin-3 Regulates Aggresome Formation of Copper-Zinc Superoxide Dismutase (SOD1) by Editing K63-linked Polyubiquitin Chains *

    doi: 10.1074/jbc.M111.299990

    Figure Lengend Snippet: Colocalization of mutant SOD1 and ataxin-3 in aggresomes. A , 293 cells were transfected with EGFP or SOD1 G85R -EGFP and treated with 10 μ m MG132 for 12 h. The supernatants of the cell lysates were immunoprecipitated with rabbit polyclonal antibodies

    Article Snippet: MG132 was purchased from Calbiochem.

    Techniques: Mutagenesis, Transfection, Immunoprecipitation

    Ataxin-3-mediated aggresome formation by mutant SOD1 depends on ataxin-3 DUB activity. A , 293 cells were co-transfected with SOD1 G85R -EGFP and HA or HA-ataxin-3, then treated with 10 μ m MG132 for 12 h. The supernatants of the cell lysates were

    Journal: The Journal of Biological Chemistry

    Article Title: Ataxin-3 Regulates Aggresome Formation of Copper-Zinc Superoxide Dismutase (SOD1) by Editing K63-linked Polyubiquitin Chains *

    doi: 10.1074/jbc.M111.299990

    Figure Lengend Snippet: Ataxin-3-mediated aggresome formation by mutant SOD1 depends on ataxin-3 DUB activity. A , 293 cells were co-transfected with SOD1 G85R -EGFP and HA or HA-ataxin-3, then treated with 10 μ m MG132 for 12 h. The supernatants of the cell lysates were

    Article Snippet: MG132 was purchased from Calbiochem.

    Techniques: Mutagenesis, Activity Assay, Transfection