mouse anti β tubulin  (Thermo Fisher)


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    Tubulin Β Ab 6 Mouse Monoclonal Antibody
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    Structured Review

    Thermo Fisher mouse anti β tubulin
    Analysis of MyD88 -dependent pathway induction. A MDDC were either untreated or treated with BpWT or BpCyaA−. Phosphorylation of p38, ERK1/2, SAP/JNK and IκB-α was determined at the indicated time-points by Western blot. A single gel was run and blotted to detect phosphorylated proteins and <t>β</t> tubulin to normalize the results. Data are from one representative out of four independent experiments performed with MDDC obtained from different donors. B MDDC were treated as in panel A, either in the absence or presence of p38 inhibitor (SB203580), ERK1/2 inhibitor (PD98059) or PI3K inhibitor (LY294002) for 48 h. Results of seven independent experiments performed with MDDC obtained from different donors are expressed as the percent of change of maturation markers (CD80, CD83, CD38) with respect to the corresponding stimulus in the absence of inhibitors. Mean ± SE of marker expression in MDDC not treated with inhibitors was for BpWT: CD80 (MFI) = 48±6; CD83 (%) = 23±6; CD38 (MFI) = 18±4; for BpCyaA−: CD80 (MFI) = 72±11; CD83 (%) = 36±7; CD38 (MFI) = 46±5. * p

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    Images

    1) Product Images from "Bordetella pertussis Commits Human Dendritic Cells to Promote a Th1/Th17 Response through the Activity of Adenylate Cyclase Toxin and MAPK-Pathways"

    Article Title: Bordetella pertussis Commits Human Dendritic Cells to Promote a Th1/Th17 Response through the Activity of Adenylate Cyclase Toxin and MAPK-Pathways

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008734

    Analysis of MyD88 -dependent pathway induction. A MDDC were either untreated or treated with BpWT or BpCyaA−. Phosphorylation of p38, ERK1/2, SAP/JNK and IκB-α was determined at the indicated time-points by Western blot. A single gel was run and blotted to detect phosphorylated proteins and β tubulin to normalize the results. Data are from one representative out of four independent experiments performed with MDDC obtained from different donors. B MDDC were treated as in panel A, either in the absence or presence of p38 inhibitor (SB203580), ERK1/2 inhibitor (PD98059) or PI3K inhibitor (LY294002) for 48 h. Results of seven independent experiments performed with MDDC obtained from different donors are expressed as the percent of change of maturation markers (CD80, CD83, CD38) with respect to the corresponding stimulus in the absence of inhibitors. Mean ± SE of marker expression in MDDC not treated with inhibitors was for BpWT: CD80 (MFI) = 48±6; CD83 (%) = 23±6; CD38 (MFI) = 18±4; for BpCyaA−: CD80 (MFI) = 72±11; CD83 (%) = 36±7; CD38 (MFI) = 46±5. * p
    Figure Legend Snippet: Analysis of MyD88 -dependent pathway induction. A MDDC were either untreated or treated with BpWT or BpCyaA−. Phosphorylation of p38, ERK1/2, SAP/JNK and IκB-α was determined at the indicated time-points by Western blot. A single gel was run and blotted to detect phosphorylated proteins and β tubulin to normalize the results. Data are from one representative out of four independent experiments performed with MDDC obtained from different donors. B MDDC were treated as in panel A, either in the absence or presence of p38 inhibitor (SB203580), ERK1/2 inhibitor (PD98059) or PI3K inhibitor (LY294002) for 48 h. Results of seven independent experiments performed with MDDC obtained from different donors are expressed as the percent of change of maturation markers (CD80, CD83, CD38) with respect to the corresponding stimulus in the absence of inhibitors. Mean ± SE of marker expression in MDDC not treated with inhibitors was for BpWT: CD80 (MFI) = 48±6; CD83 (%) = 23±6; CD38 (MFI) = 18±4; for BpCyaA−: CD80 (MFI) = 72±11; CD83 (%) = 36±7; CD38 (MFI) = 46±5. * p

    Techniques Used: Western Blot, Marker, Expressing

    2) Product Images from "Alpha-synuclein aggresomes inhibit ciliogenesis and multiple functions of the centrosome"

    Article Title: Alpha-synuclein aggresomes inhibit ciliogenesis and multiple functions of the centrosome

    Journal: Biology Open

    doi: 10.1242/bio.054338

    Olfactory cilia in zebrafish larvae are severely reduced in the presence of aggresomes. (A) The neuronal dopaminergic network in 3 dpf zebrafish forebrain viewed from the dorsal aspect, detected by TH-staining (green). Acetylated tubulin (red) stains axon tracts and cilia. (B–E) Overexpression of control GFP or α-syn, α-synA30P, α-synA53T familial mutations does not cause any anatomical defects in zebrafish larvae. (F) By 3 dpf extensive numbers of cilia are visible at the olfactory pit. (G–I) Overexpression of any of the three forms of α-syn (wild type, A30P or A53T) severely reduces numbers of cilia in the olfactory pit. Cilia length is also reduced. (J,K) Embryos treated with MG132 showed an extensive reduction in number of cilia. (L) Quantification of cilia numbers from above experiments ( P =0.001, by one-way ANOVA, n =3). Cilia were counted from the confocal generated z-stacks, projected images of which are shown in this figure. (M) Quantification of length of cilia from F-I ( P =0.0088, by one-way ANOVA, n =3). Scale bars: 100 μM.
    Figure Legend Snippet: Olfactory cilia in zebrafish larvae are severely reduced in the presence of aggresomes. (A) The neuronal dopaminergic network in 3 dpf zebrafish forebrain viewed from the dorsal aspect, detected by TH-staining (green). Acetylated tubulin (red) stains axon tracts and cilia. (B–E) Overexpression of control GFP or α-syn, α-synA30P, α-synA53T familial mutations does not cause any anatomical defects in zebrafish larvae. (F) By 3 dpf extensive numbers of cilia are visible at the olfactory pit. (G–I) Overexpression of any of the three forms of α-syn (wild type, A30P or A53T) severely reduces numbers of cilia in the olfactory pit. Cilia length is also reduced. (J,K) Embryos treated with MG132 showed an extensive reduction in number of cilia. (L) Quantification of cilia numbers from above experiments ( P =0.001, by one-way ANOVA, n =3). Cilia were counted from the confocal generated z-stacks, projected images of which are shown in this figure. (M) Quantification of length of cilia from F-I ( P =0.0088, by one-way ANOVA, n =3). Scale bars: 100 μM.

    Techniques Used: Staining, Over Expression, Generated

    Aggresomes can be formed in cells by overexpression of alpha synuclein or MG132 treatment. (A,A′) Vimentin (red) in control HeLa cells forms a fibrous network around the nuclei (DAPI, blue), which is unaffected by the expression of the GFP. (B,B′) When expressing GFP- α-syn, vimentin-positive aggresomes appear in cells, juxtaposed to the nucleus. Similar results are obtained with GFP-α-synA30P (C,C′) and GFP-α-synA53T mutants (D,D′) and in SH-SY5Y cells treated in the same way (E–H). (I–L) Aggresomes can also be induced by treatment with MG132. (I,J) The vimentin distribution changes from a filamentous pattern around the nuclei to caging the aggresome. (K,L) γ-tubulin (red) staining the centrosomes as two punctae in control cells (K). Following MG132 treatment, γ-tubulin forms a condensed structure around the aggresome (L). (M–P) The expression pattern of endogenous α-syn was also investigated to determine whether this protein co-localises within the aggresome. (M,M′) In control cells, endogenous α-syn (green) staining was widespread and diffuse within the cytoplasm with vimentin (red) forming a filamentous network. (N,N′) Following MG132 treatment, α-syn aggregates were observed and co-localised with vimentin staining within the aggresomes. (O,O′) In control cells, γ-tubulin (red) was observed as two punctae with α-syn diffuse within the cytoplasm. (P,P′) γ-tubulin staining (red) also co-localises with endogenous α-syn in the aggresome when treated with MG132. (Q,Q′) Differentiated SH-SY5Y cells were mock-treated and vimentin (red) staining was observed surrounding the nuclei as well as along the axon. (R,R′) In cells treated with MG132 (1 μM for 18 h) vimentin staining changed to a compact structure near the nucleus, indicative of aggresomes. (S) In mock-treated cells, γ-tubulin formed two punctae next to the nucleus. (T) In MG132 treated cells, aggresomes were detected by γ-tubulin staining. Differentiated SH-SY5Y cells are TH positive (green). (U,U′) In rat basal ganglion neurons, vimentin staining (red) is abundant around the nuclei and along the axon. (V,V′) When treated with MG132, vimentin localises to the aggresome. (W) In rat basal ganglion neurons, the γ-tubulin is observed at two punctae close to the nucleus. (X) Upon MG132 treatment, the γ-tubulin staining now forms a larger structure next to the nucleus. (Y) In untreated RPE1-hTERT cells, γ-tubulin stains the centrosome. (Z) Upon MG132 treatment it stains the aggresome. (α,β) Similar results are obtained with MEFs. Scale bars: 10 μM. DNA/nuclei stained with DAPI (blue) where indicated.
    Figure Legend Snippet: Aggresomes can be formed in cells by overexpression of alpha synuclein or MG132 treatment. (A,A′) Vimentin (red) in control HeLa cells forms a fibrous network around the nuclei (DAPI, blue), which is unaffected by the expression of the GFP. (B,B′) When expressing GFP- α-syn, vimentin-positive aggresomes appear in cells, juxtaposed to the nucleus. Similar results are obtained with GFP-α-synA30P (C,C′) and GFP-α-synA53T mutants (D,D′) and in SH-SY5Y cells treated in the same way (E–H). (I–L) Aggresomes can also be induced by treatment with MG132. (I,J) The vimentin distribution changes from a filamentous pattern around the nuclei to caging the aggresome. (K,L) γ-tubulin (red) staining the centrosomes as two punctae in control cells (K). Following MG132 treatment, γ-tubulin forms a condensed structure around the aggresome (L). (M–P) The expression pattern of endogenous α-syn was also investigated to determine whether this protein co-localises within the aggresome. (M,M′) In control cells, endogenous α-syn (green) staining was widespread and diffuse within the cytoplasm with vimentin (red) forming a filamentous network. (N,N′) Following MG132 treatment, α-syn aggregates were observed and co-localised with vimentin staining within the aggresomes. (O,O′) In control cells, γ-tubulin (red) was observed as two punctae with α-syn diffuse within the cytoplasm. (P,P′) γ-tubulin staining (red) also co-localises with endogenous α-syn in the aggresome when treated with MG132. (Q,Q′) Differentiated SH-SY5Y cells were mock-treated and vimentin (red) staining was observed surrounding the nuclei as well as along the axon. (R,R′) In cells treated with MG132 (1 μM for 18 h) vimentin staining changed to a compact structure near the nucleus, indicative of aggresomes. (S) In mock-treated cells, γ-tubulin formed two punctae next to the nucleus. (T) In MG132 treated cells, aggresomes were detected by γ-tubulin staining. Differentiated SH-SY5Y cells are TH positive (green). (U,U′) In rat basal ganglion neurons, vimentin staining (red) is abundant around the nuclei and along the axon. (V,V′) When treated with MG132, vimentin localises to the aggresome. (W) In rat basal ganglion neurons, the γ-tubulin is observed at two punctae close to the nucleus. (X) Upon MG132 treatment, the γ-tubulin staining now forms a larger structure next to the nucleus. (Y) In untreated RPE1-hTERT cells, γ-tubulin stains the centrosome. (Z) Upon MG132 treatment it stains the aggresome. (α,β) Similar results are obtained with MEFs. Scale bars: 10 μM. DNA/nuclei stained with DAPI (blue) where indicated.

    Techniques Used: Over Expression, Expressing, Staining

    Aggresomes inhibit ciliogenesis. All panels: DNA/nuclei stained with DAPI (blue), green label is indicated in each panel, cilia (white arrows) can be identified by acetylated tubulin (red). (A–F) When transfected with a GFP-α-syn expression plasmid (α-syn and A30P and A53T familial mutants) or treated with MG132, cilia formation is inhibited in undifferentiated SH-SH5Y cells (B, C and D versus A, F versus E). In the presence of aggresomes differentiated SH-SY5Y cells are no longer able to form cilia (H versus G). When treated with MG132, TH-positive basal ganglion neurons are no longer able to form cilia (J versus I). The acetylated tubulin signal (red) in J and I is overexposed to ensure no cilia were missed. (K) Quantification of ciliation: GFP expressing versus GFP-α-syn expression, ( P =0.0003, by one-way ANOVA, 100 cells, n =3); undifferentiated SH-SY5Y, untreated versus MG132, ( P =0.0001, by Student's t -test, 100 cell, n =3); differentiated SH-SY5Y, untreated versus MG132, ( P =0.0001 by Student's t -test, 100 cells counted, n =3). Scale bars: 10 μM.
    Figure Legend Snippet: Aggresomes inhibit ciliogenesis. All panels: DNA/nuclei stained with DAPI (blue), green label is indicated in each panel, cilia (white arrows) can be identified by acetylated tubulin (red). (A–F) When transfected with a GFP-α-syn expression plasmid (α-syn and A30P and A53T familial mutants) or treated with MG132, cilia formation is inhibited in undifferentiated SH-SH5Y cells (B, C and D versus A, F versus E). In the presence of aggresomes differentiated SH-SY5Y cells are no longer able to form cilia (H versus G). When treated with MG132, TH-positive basal ganglion neurons are no longer able to form cilia (J versus I). The acetylated tubulin signal (red) in J and I is overexposed to ensure no cilia were missed. (K) Quantification of ciliation: GFP expressing versus GFP-α-syn expression, ( P =0.0003, by one-way ANOVA, 100 cells, n =3); undifferentiated SH-SY5Y, untreated versus MG132, ( P =0.0001, by Student's t -test, 100 cell, n =3); differentiated SH-SY5Y, untreated versus MG132, ( P =0.0001 by Student's t -test, 100 cells counted, n =3). Scale bars: 10 μM.

    Techniques Used: Staining, Transfection, Expressing, Plasmid Preparation

    3) Product Images from "EWS-FLI-1 creates a cell surface microenvironment conducive to IGF signaling by inducing pappalysin-1"

    Article Title: EWS-FLI-1 creates a cell surface microenvironment conducive to IGF signaling by inducing pappalysin-1

    Journal: Genes & Cancer

    doi: 10.18632/genesandcancer.159

    Pappalysin-1 silencing results in accumulation of IGFBPs and reduced IGF signaling in Ewing sarcoma cells (A) Pappalysin-1 expression was silenced by siRNA transfection in four Ewing sarcoma cell lines (A673, EW8, TC32, and TC71). Immunoblotting was performed using antibodies against IGFBP2, IGFBP4, IGFBP5, PGK1, pappalysin-1, IGF-1R, phospho-IGF-1R (phosphorylated at tyrosine 1135), phospho-Akt (phosphorylated at serine 473), and tubulin. (B) EWS-FLI-1 silencing recapitulates the pappalysin-1 silencing effects in Ewing sarcoma. EWS-FLI-1 expression was silenced by an shRNA targeting the C-terminal region of FLI- 1 in A673 and EW8 cells. Immunoblotting was performed using antibodies against IGFBP2, IGFBP4, IGFBP5, PGK1, pappalysin-1, FLI-1 (C-terminus), IGF-1R, phospho-IGF-1R (phosphorylated at tyrosine 1135), phospho-Akt (phosphorylated at serine 473), and tubulin. (C) Expression of pappalysin-1 inhibitors, stanniocalcin 1 and 2 (STC1 and STC2), recapitulates the pappalysin-1 silencing effects in Ewing sarcoma. C-terminally FLAG-tagged STC1 and STC2 were stably expressed in A673 cells by lentiviral infection and puromycin selection. Immunoblotting was performed using antibodies against IGFBP2, IGFBP4, IGFBP5, PGK1, pappalysin-1, IGF-1R, phospho-IGF-1R (phosphorylated at tyrosine 1135), phospho-Akt (phosphorylated at serine 473), and tubulin.
    Figure Legend Snippet: Pappalysin-1 silencing results in accumulation of IGFBPs and reduced IGF signaling in Ewing sarcoma cells (A) Pappalysin-1 expression was silenced by siRNA transfection in four Ewing sarcoma cell lines (A673, EW8, TC32, and TC71). Immunoblotting was performed using antibodies against IGFBP2, IGFBP4, IGFBP5, PGK1, pappalysin-1, IGF-1R, phospho-IGF-1R (phosphorylated at tyrosine 1135), phospho-Akt (phosphorylated at serine 473), and tubulin. (B) EWS-FLI-1 silencing recapitulates the pappalysin-1 silencing effects in Ewing sarcoma. EWS-FLI-1 expression was silenced by an shRNA targeting the C-terminal region of FLI- 1 in A673 and EW8 cells. Immunoblotting was performed using antibodies against IGFBP2, IGFBP4, IGFBP5, PGK1, pappalysin-1, FLI-1 (C-terminus), IGF-1R, phospho-IGF-1R (phosphorylated at tyrosine 1135), phospho-Akt (phosphorylated at serine 473), and tubulin. (C) Expression of pappalysin-1 inhibitors, stanniocalcin 1 and 2 (STC1 and STC2), recapitulates the pappalysin-1 silencing effects in Ewing sarcoma. C-terminally FLAG-tagged STC1 and STC2 were stably expressed in A673 cells by lentiviral infection and puromycin selection. Immunoblotting was performed using antibodies against IGFBP2, IGFBP4, IGFBP5, PGK1, pappalysin-1, IGF-1R, phospho-IGF-1R (phosphorylated at tyrosine 1135), phospho-Akt (phosphorylated at serine 473), and tubulin.

    Techniques Used: Expressing, Transfection, shRNA, Stable Transfection, Infection, Selection

    EWS-FLI-1 induces pappalysin-1 expression (A) shRNA-mediated silencing of EWS-FLI-1 in A673 Ewing sarcoma cells. A673 cells were infected with lentiviruses expressing an shRNA against FLI-1 C-terminal region or luciferase (control) and were selected with 2 µg/ml puromycin for 2 days. Whole cell lysates were prepared and immunoblotting was performed using antibodies against FLI-1 C-terminus and tubulin (loading control). (B) Reduced pappalysin-1 protein levels in A673 cell secretome upon EWS-FLI-1 silencing.(C) EWS-FLI-1 silencing in A673 cells results in reduced pappalysin-1 transcript levels. EWS-FLI-1 was silenced as in (A) and the pappalysin-1 mRNA levels were examined by real-time PCR. An asterisk denotes p
    Figure Legend Snippet: EWS-FLI-1 induces pappalysin-1 expression (A) shRNA-mediated silencing of EWS-FLI-1 in A673 Ewing sarcoma cells. A673 cells were infected with lentiviruses expressing an shRNA against FLI-1 C-terminal region or luciferase (control) and were selected with 2 µg/ml puromycin for 2 days. Whole cell lysates were prepared and immunoblotting was performed using antibodies against FLI-1 C-terminus and tubulin (loading control). (B) Reduced pappalysin-1 protein levels in A673 cell secretome upon EWS-FLI-1 silencing.(C) EWS-FLI-1 silencing in A673 cells results in reduced pappalysin-1 transcript levels. EWS-FLI-1 was silenced as in (A) and the pappalysin-1 mRNA levels were examined by real-time PCR. An asterisk denotes p

    Techniques Used: Expressing, shRNA, Infection, Luciferase, Real-time Polymerase Chain Reaction

    4) Product Images from "Proteomic Analysis of the EWS-Fli-1 Interactome Reveals the Role of the Lysosome in EWS-Fli-1 Turnover"

    Article Title: Proteomic Analysis of the EWS-Fli-1 Interactome Reveals the Role of the Lysosome in EWS-Fli-1 Turnover

    Journal: Journal of Proteome Research

    doi: 10.1021/pr500387m

    EWS-Fli-1 turns over by a lysosome-dependent mechanism: (A) Knockdown of CIMPR or VPS26A results in destabilization of FLAG-EWS-Fli-1. 293 cells were cotransfected with FLAG-EWS-Fli-1 and shRNA against luciferase (control), CIMPR, or VPS26A. Forty-eight hours after transfection, the levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting. Nucleolin serves as a loading control. (B) TFEB induces EWS-Fli-1 degradation in 293 cells. 293 cells were cotransfected with FLAG-EWS-Fli-1 and HA-TFEB or empty vector. Forty-eight hours after transfection, the levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting. Tubulin serves as a loading control. (C) Chloroquine stabilizes EWS-Fli-1 in 293 cells. 293 cells were transfected with FLAG-EWS-Fli-1. Transfected cells were left untreated (control) or treated with 100 μM chloroquine for 12 h. The levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting. Tubulin serves as a loading control. (D) Cathepsin D degrades EWS-Fli-1, but not p53, in 293 cells. 293 cells were cotransfected with FLAG-EWS-Fli-1 and cathepsin D or empty vector. Transfected cells were left untreated or treated with 100 μM chloroquine for 12 h or 100 nM pepstatin A for 12 h. 293 cells were cotransfected with FLAG-p53 and cathepsin D or empty vector. The levels of FLAG-EWS-Fli-1 and FLAG-p53 were examined by anti-FLAG immunoblotting. Nucleolin serves as a loading control. (E) Cathepsin D degrades endogenous EWS-Fli-1 in A673 Ewing sarcoma cells. A673 cells were infected with a lentivirus vector expressing cathepsin D or an empty vector, the infected cells were selected with puromycin, and the levels of endogenous EWS-Fli-1 were examined by anti-Fli-1 C-terminus antibody immunoblotting at 4 days after infection. Nucleolin serves as a loading control. (F) Chloroquine stabilizes endogenous EWS-Fli-1 in A673 cells. A673 cells were left untreated, treated with 100 μM chloroquine for 12 h, or treated with 10 μM MG-132 for 12 h. The levels of EWS-Fli-1 were examined by anti-Fli-1 C-terminus immunoblotting. While chloroquine increased the levels of endogenous EWS-Fli-1, MG-132 had no effect on the EWS-Fli-1 protein levels, suggesting that EWS-Fli-1 turns over by a lysosomal, but not proteasomal mechanism. (G) Endogenous EWS-Fli-1 in A673 cells displays increased lysosomal location upon chloroquine treatment. A673 cells were treated with 100 μM chloroquine for 12 h or left untreated, and the whole cell extract (WCE) and lysosomal fraction were isolated. The abundance of EWS-Fli-1, LAMP2, p62/SQSTM1, and mSin3A in each fraction was determined by immunoblotting.
    Figure Legend Snippet: EWS-Fli-1 turns over by a lysosome-dependent mechanism: (A) Knockdown of CIMPR or VPS26A results in destabilization of FLAG-EWS-Fli-1. 293 cells were cotransfected with FLAG-EWS-Fli-1 and shRNA against luciferase (control), CIMPR, or VPS26A. Forty-eight hours after transfection, the levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting. Nucleolin serves as a loading control. (B) TFEB induces EWS-Fli-1 degradation in 293 cells. 293 cells were cotransfected with FLAG-EWS-Fli-1 and HA-TFEB or empty vector. Forty-eight hours after transfection, the levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting. Tubulin serves as a loading control. (C) Chloroquine stabilizes EWS-Fli-1 in 293 cells. 293 cells were transfected with FLAG-EWS-Fli-1. Transfected cells were left untreated (control) or treated with 100 μM chloroquine for 12 h. The levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting. Tubulin serves as a loading control. (D) Cathepsin D degrades EWS-Fli-1, but not p53, in 293 cells. 293 cells were cotransfected with FLAG-EWS-Fli-1 and cathepsin D or empty vector. Transfected cells were left untreated or treated with 100 μM chloroquine for 12 h or 100 nM pepstatin A for 12 h. 293 cells were cotransfected with FLAG-p53 and cathepsin D or empty vector. The levels of FLAG-EWS-Fli-1 and FLAG-p53 were examined by anti-FLAG immunoblotting. Nucleolin serves as a loading control. (E) Cathepsin D degrades endogenous EWS-Fli-1 in A673 Ewing sarcoma cells. A673 cells were infected with a lentivirus vector expressing cathepsin D or an empty vector, the infected cells were selected with puromycin, and the levels of endogenous EWS-Fli-1 were examined by anti-Fli-1 C-terminus antibody immunoblotting at 4 days after infection. Nucleolin serves as a loading control. (F) Chloroquine stabilizes endogenous EWS-Fli-1 in A673 cells. A673 cells were left untreated, treated with 100 μM chloroquine for 12 h, or treated with 10 μM MG-132 for 12 h. The levels of EWS-Fli-1 were examined by anti-Fli-1 C-terminus immunoblotting. While chloroquine increased the levels of endogenous EWS-Fli-1, MG-132 had no effect on the EWS-Fli-1 protein levels, suggesting that EWS-Fli-1 turns over by a lysosomal, but not proteasomal mechanism. (G) Endogenous EWS-Fli-1 in A673 cells displays increased lysosomal location upon chloroquine treatment. A673 cells were treated with 100 μM chloroquine for 12 h or left untreated, and the whole cell extract (WCE) and lysosomal fraction were isolated. The abundance of EWS-Fli-1, LAMP2, p62/SQSTM1, and mSin3A in each fraction was determined by immunoblotting.

    Techniques Used: shRNA, Luciferase, Transfection, Plasmid Preparation, Infection, Expressing, Isolation

    Tandem affinity purification analysis of the EWS-Fli-1-interacting proteins. (A) Tandem affinity purification procedure. 293T cells were transfected with FLAG-His-EWS-Fli-1. Forty-eight hours after transfection, FLAG-His-EWS-Fli-1 and its interacting proteins were isolated by nickel affinity chromatography followed by anti-FLAG immunoprecipitation and the protein sample was analyzed by tandem mass spectrometry. (B) EWS peptides assigned with high confidence. (C) RNA helicase A peptides assigned with high confidence. (D) FLAG-His-EWS-Fli-1 is mostly insoluble under the lysis conditions used for tandem affinity purification. The abundance of FLAG-His-EWS-Fli-1 in whole cell lysate (lane 1 and 4), tandem affinity purification lysate (lane 2 and 5), and postlysis pellet (lane 3 and 6) was determined by anti-FLAG immunoblotting. Tubulin serves as a loading control.
    Figure Legend Snippet: Tandem affinity purification analysis of the EWS-Fli-1-interacting proteins. (A) Tandem affinity purification procedure. 293T cells were transfected with FLAG-His-EWS-Fli-1. Forty-eight hours after transfection, FLAG-His-EWS-Fli-1 and its interacting proteins were isolated by nickel affinity chromatography followed by anti-FLAG immunoprecipitation and the protein sample was analyzed by tandem mass spectrometry. (B) EWS peptides assigned with high confidence. (C) RNA helicase A peptides assigned with high confidence. (D) FLAG-His-EWS-Fli-1 is mostly insoluble under the lysis conditions used for tandem affinity purification. The abundance of FLAG-His-EWS-Fli-1 in whole cell lysate (lane 1 and 4), tandem affinity purification lysate (lane 2 and 5), and postlysis pellet (lane 3 and 6) was determined by anti-FLAG immunoblotting. Tubulin serves as a loading control.

    Techniques Used: Affinity Purification, Transfection, Isolation, Affinity Chromatography, Immunoprecipitation, Mass Spectrometry, Lysis

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    Staining:

    Article Title: Characterization of pediatric cystic fibrosis airway epithelial cell cultures at the air-liquid interface obtained by non-invasive nasal cytology brush sampling
    Article Snippet: The cells were then incubated with the primary antibody or dye in staining buffer for one hour at room temperature. .. Cells were stained with Phalloidin Rhodamine (1:100; Invitrogen, Switzerland), rabbit anti-MUC5AC (clone H-160, 1:100, Santa Cruz Biotechnologies, US) mouse anti-β-tubulin (clone 2 28 33, 1:100, Invitrogen, Switzerland), rabbit anti-p63 (clone EPR5701, 1:60, Abcam, UK), rabbit anti-ZO-1 (Zona occludens-1; ab59720, 1:50, Abcam, UK), and mouse anti-CFTR (clone 24–1, 1:100, R & D Systems, UK). .. After washing with staining buffer, the cells were incubated for one hour with the secondary antibody in staining buffer at room temperature: anti-mouse Alexa Fluor 488 (Invitrogen, US) and anti-rabbit Alexa Fluor 647 (Invitrogen, US).

    Blocking Assay:

    Article Title: Autologous skeletal muscle derived cells expressing a novel functional dystrophin provide a potential therapy for Duchenne Muscular Dystrophy
    Article Snippet: .. The membrane was then blocked with Odyssey block solution (LI-COR Biosciences, Cambridge, UK) for 60min, incubated by a cocktail of primary antibodies of rabbit anti-GFP antibody (1:2000, Invitrogen, Paisley, UK) (or rabbit anti-dystrophin antibody, 1:2000, Fisher Scientific, Loughborough, UK) and mouse anti-tubulin (1:2000, Invitrogen, Paisley, UK) overnight at 4 °C. .. After washing with PBS containing 1% Tween 20 (PBST) for 15 min × 3 times at room temperature, the membrane was then incubated with IRDye 680 RD goat anti-rabbit and IRDye 800CW goat anti-mouse 2nd antibodies (1:15000, LI-COR Biosciences, Cambridge, UK) for 1–2 hrs at RT.

    other:

    Article Title: Proteomic Analysis of the EWS-Fli-1 Interactome Reveals the Role of the Lysosome in EWS-Fli-1 Turnover
    Article Snippet: Immunoblotting Immunoblotting was performed as described., The following antibodies were used: rabbit polyclonal anti-CIMPR (ab32815, Abcam); mouse monoclonal anti-cyclin D1 (2926, Cell Signaling Technologies); mouse monoclonal anti-FLAG (M2, Sigma-Aldrich); rabbit polyclonal anti-FLAG (Immunology Consultants Laboratory, Inc.); rabbit polyclonal anti-Fli-1 (ab15289, Abcam); mouse monoclonal anti-HA (16B12, Covance); mouse monoclonal anti-LAMP2 (55803, BD Biosciences); rabbit polyclonal anti-mSin3A (K-20, Santa Cruz Biotechnology); rabbit polyclonal anti-Myc (N262, Santa Cruz Biotechnology); mouse monoclonal anti-nucleolin (C23, Santa Cruz Biotechnology); mouse monoclonal anti-p62/SQSTM1 (610832, BD Biosciences); and mouse monoclonal anti-tubulin (DM1A, Lab Vision).

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  • 94
    Thermo Fisher mouse anti β tubulin
    Analysis of MyD88 -dependent pathway induction. A MDDC were either untreated or treated with BpWT or BpCyaA−. Phosphorylation of p38, ERK1/2, SAP/JNK and IκB-α was determined at the indicated time-points by Western blot. A single gel was run and blotted to detect phosphorylated proteins and <t>β</t> tubulin to normalize the results. Data are from one representative out of four independent experiments performed with MDDC obtained from different donors. B MDDC were treated as in panel A, either in the absence or presence of p38 inhibitor (SB203580), ERK1/2 inhibitor (PD98059) or PI3K inhibitor (LY294002) for 48 h. Results of seven independent experiments performed with MDDC obtained from different donors are expressed as the percent of change of maturation markers (CD80, CD83, CD38) with respect to the corresponding stimulus in the absence of inhibitors. Mean ± SE of marker expression in MDDC not treated with inhibitors was for BpWT: CD80 (MFI) = 48±6; CD83 (%) = 23±6; CD38 (MFI) = 18±4; for BpCyaA−: CD80 (MFI) = 72±11; CD83 (%) = 36±7; CD38 (MFI) = 46±5. * p
    Mouse Anti β Tubulin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti β tubulin/product/Thermo Fisher
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti β tubulin - by Bioz Stars, 2021-06
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    94
    Thermo Fisher anti tubulin
    Phosphorylation at Ser-41 is required for plasma membrane association of GAP43. (A) High-resolution images of single focal planes of living PC12 cells expressing the constructs as indicated. Photoactivation was performed in the whole field of view before imaging. Right, quantitation of peripheral enrichment by densitometric analysis of single z -stack images (for details see Materials and Methods ). Mean ± SEM, n = 29–35 cells from three of four independent experiments. ***Significantly different values compared with GAP43 wt . Note that GAP43 S41D -PAGFP exhibits higher and GAP43 S41A -PAGFP lower peripheral enrichment than wild-type GAP43-PAGFP. Arrowheads indicate examples for obvious enrichment of GAP43 S41A -PAGFP at the periphery. Scale bar, 10 μm. (B) Schematic representation of the plasma membrane fractionation assay to analyze the interaction of GAP43 with the neural plasma membrane (left). Immunoblots show the distribution of PAGFP-tagged GAP43 constructs (GFP), <t>tubulin</t> (tub), and transferrin receptor (TFR) in the cytosolic (cyt), membrane wash (mw), and plasma membrane (pm) fractions. Note that GAP43 wt is present in the cytosolic and pm fractions, whereas GAP43 S41A is mainly present in the cytosol and GAP43 S41D in the pm fraction. Detection of GAP43-PAGFP and endogenous GAP43 with a Ser-41 phosphorylation-specific antibody (anti-phosphoneuromodulin) indicates that phosphorylated GAP43 is mainly present in the pm fraction. (C) Image of a single focal plane of a living PC12 cell expressing PAGFP-F (top) and immunoblot showing the distribution of PAGFP-F (GFP), tubulin (tub), and transferrin receptor (TFR) in the indicated fractions. Note the high enrichment of PAGFP-F in the pm fraction. Scale bar, 10 μm. Numbers to the sides of the gel blots indicate molecular mass standards in kilodaltons (B, C). Statistical analysis was performed using Student's t test. ***, p
    Anti Tubulin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti tubulin/product/Thermo Fisher
    Average 94 stars, based on 1 article reviews
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    94
    Thermo Fisher tubulin α ab 2 mouse monoclonal antibody
    Silencing of integrin β1 inhibits IGFBP3-induced migration A. The activities of Cdc42, Rac1 and RhoA were detected in IGBBP3 knockdown cells (IGFBP3 sh4 and sh5) and the corresponding controls (pLKO-GFP). Equal amounts of input protein were subjected to Western blot using anti-Cdc42 (total Cdc42), anti-Rac1 (total Rac1), and anti-RhoA (total RhoA) antibodies. Equal amounts of protein were incubated with GST-PAK1 (detection of active Cdc42 or Rac1) and GST-Rhotekin (detection of active RhoA). Complexes were collected with gluthathione-Sepharose and resolved by Western blot. GTPγS was served as a positive control. Anti-GST antibodies were served as a loading control. B. The density of each band was measured by image J and normalized with the controls (LN1–1 pLKO-GFP). The activities of active small GTPase were conducted by dividing the density of active small GTPase to that of GST loading controls. The relative activities were obtained when the activity of active small GTPase in LN1–1 pLKO-GFP were set to 1. C. Representative data shows the relative migration activity of OEC-M1 cells with dominant-negative Cdc42 (Cdc42dn) and the corresponding controls (PB). The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 PB treated with treated with IGFBP3 and PB-Cdc42dn cells with/without IGFBP3 treatment with that in OEC-M1 PB cells. D. Representative data shows the relative migration activity of IGFBP3 expressing cells (OEC-M1 IGFBP3) and the corresponding controls (OEC-M1 PB) with anti-integrin β1 (200 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 PB and IGFBP3 cells treated with anti-integrin β1 or OEC-M1 IGFBP3 cells treated with IgG antibodies (200 ng/ml) to that in OEC-M1 PB cells treated with IgG antibodies. E. Immunoblot analysis of integrin β1 protein in OEC-M1 cells with ITGB1 shRNA expression (OEC-M1 ITGB1 sh3 and sh4) and vector controls (OEC-M1 pLKO-GFP). <t>α-tubulin</t> serves as an internal control. F. Representative data shows the relative migration activity of OEC-M1 pLKO-GFP, ITGB1 sh3 and sh4 cells upon IGFBP3 (100 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 with ITGB1 knockdown and IGFBP3 treatment with that in the control cells. G. Representative data showed the relative migration activity of OEC-M1 treated with 10, 20 uM of PD98059 (PD) and dimethyl sulfoxide (DMSO) upon IGFBP3 (100 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell/per field in OEC-M1 treated with PD98059 and IGFBP3 with that in the control cells. H. Immunoblot analysis revealed knockdown of ITGB1 inhibited IGFBP3-induced ERK phosphorylation at different time points in OEC-M1 cells (upper panel, 0, untreated; 10, 10 min; 30, 30 min for 100 ng/ml IGFBP3 treatment). The ratio of phosphorylated ERK/total ERK was obtained by dividing the intensity of phosphorylated ERK to that of total ERK. The relative expression was obtained when the ration of untreated cells were set to 1 (lower panel). Bar, SE; ** p
    Tubulin α Ab 2 Mouse Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Analysis of MyD88 -dependent pathway induction. A MDDC were either untreated or treated with BpWT or BpCyaA−. Phosphorylation of p38, ERK1/2, SAP/JNK and IκB-α was determined at the indicated time-points by Western blot. A single gel was run and blotted to detect phosphorylated proteins and β tubulin to normalize the results. Data are from one representative out of four independent experiments performed with MDDC obtained from different donors. B MDDC were treated as in panel A, either in the absence or presence of p38 inhibitor (SB203580), ERK1/2 inhibitor (PD98059) or PI3K inhibitor (LY294002) for 48 h. Results of seven independent experiments performed with MDDC obtained from different donors are expressed as the percent of change of maturation markers (CD80, CD83, CD38) with respect to the corresponding stimulus in the absence of inhibitors. Mean ± SE of marker expression in MDDC not treated with inhibitors was for BpWT: CD80 (MFI) = 48±6; CD83 (%) = 23±6; CD38 (MFI) = 18±4; for BpCyaA−: CD80 (MFI) = 72±11; CD83 (%) = 36±7; CD38 (MFI) = 46±5. * p

    Journal: PLoS ONE

    Article Title: Bordetella pertussis Commits Human Dendritic Cells to Promote a Th1/Th17 Response through the Activity of Adenylate Cyclase Toxin and MAPK-Pathways

    doi: 10.1371/journal.pone.0008734

    Figure Lengend Snippet: Analysis of MyD88 -dependent pathway induction. A MDDC were either untreated or treated with BpWT or BpCyaA−. Phosphorylation of p38, ERK1/2, SAP/JNK and IκB-α was determined at the indicated time-points by Western blot. A single gel was run and blotted to detect phosphorylated proteins and β tubulin to normalize the results. Data are from one representative out of four independent experiments performed with MDDC obtained from different donors. B MDDC were treated as in panel A, either in the absence or presence of p38 inhibitor (SB203580), ERK1/2 inhibitor (PD98059) or PI3K inhibitor (LY294002) for 48 h. Results of seven independent experiments performed with MDDC obtained from different donors are expressed as the percent of change of maturation markers (CD80, CD83, CD38) with respect to the corresponding stimulus in the absence of inhibitors. Mean ± SE of marker expression in MDDC not treated with inhibitors was for BpWT: CD80 (MFI) = 48±6; CD83 (%) = 23±6; CD38 (MFI) = 18±4; for BpCyaA−: CD80 (MFI) = 72±11; CD83 (%) = 36±7; CD38 (MFI) = 46±5. * p

    Article Snippet: Mouse anti-β tubulin was from Invitrogen (Paisley, UK).

    Techniques: Western Blot, Marker, Expressing

    Phosphorylation at Ser-41 is required for plasma membrane association of GAP43. (A) High-resolution images of single focal planes of living PC12 cells expressing the constructs as indicated. Photoactivation was performed in the whole field of view before imaging. Right, quantitation of peripheral enrichment by densitometric analysis of single z -stack images (for details see Materials and Methods ). Mean ± SEM, n = 29–35 cells from three of four independent experiments. ***Significantly different values compared with GAP43 wt . Note that GAP43 S41D -PAGFP exhibits higher and GAP43 S41A -PAGFP lower peripheral enrichment than wild-type GAP43-PAGFP. Arrowheads indicate examples for obvious enrichment of GAP43 S41A -PAGFP at the periphery. Scale bar, 10 μm. (B) Schematic representation of the plasma membrane fractionation assay to analyze the interaction of GAP43 with the neural plasma membrane (left). Immunoblots show the distribution of PAGFP-tagged GAP43 constructs (GFP), tubulin (tub), and transferrin receptor (TFR) in the cytosolic (cyt), membrane wash (mw), and plasma membrane (pm) fractions. Note that GAP43 wt is present in the cytosolic and pm fractions, whereas GAP43 S41A is mainly present in the cytosol and GAP43 S41D in the pm fraction. Detection of GAP43-PAGFP and endogenous GAP43 with a Ser-41 phosphorylation-specific antibody (anti-phosphoneuromodulin) indicates that phosphorylated GAP43 is mainly present in the pm fraction. (C) Image of a single focal plane of a living PC12 cell expressing PAGFP-F (top) and immunoblot showing the distribution of PAGFP-F (GFP), tubulin (tub), and transferrin receptor (TFR) in the indicated fractions. Note the high enrichment of PAGFP-F in the pm fraction. Scale bar, 10 μm. Numbers to the sides of the gel blots indicate molecular mass standards in kilodaltons (B, C). Statistical analysis was performed using Student's t test. ***, p

    Journal: Molecular Biology of the Cell

    Article Title: Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43

    doi: 10.1091/mbc.E13-12-0737

    Figure Lengend Snippet: Phosphorylation at Ser-41 is required for plasma membrane association of GAP43. (A) High-resolution images of single focal planes of living PC12 cells expressing the constructs as indicated. Photoactivation was performed in the whole field of view before imaging. Right, quantitation of peripheral enrichment by densitometric analysis of single z -stack images (for details see Materials and Methods ). Mean ± SEM, n = 29–35 cells from three of four independent experiments. ***Significantly different values compared with GAP43 wt . Note that GAP43 S41D -PAGFP exhibits higher and GAP43 S41A -PAGFP lower peripheral enrichment than wild-type GAP43-PAGFP. Arrowheads indicate examples for obvious enrichment of GAP43 S41A -PAGFP at the periphery. Scale bar, 10 μm. (B) Schematic representation of the plasma membrane fractionation assay to analyze the interaction of GAP43 with the neural plasma membrane (left). Immunoblots show the distribution of PAGFP-tagged GAP43 constructs (GFP), tubulin (tub), and transferrin receptor (TFR) in the cytosolic (cyt), membrane wash (mw), and plasma membrane (pm) fractions. Note that GAP43 wt is present in the cytosolic and pm fractions, whereas GAP43 S41A is mainly present in the cytosol and GAP43 S41D in the pm fraction. Detection of GAP43-PAGFP and endogenous GAP43 with a Ser-41 phosphorylation-specific antibody (anti-phosphoneuromodulin) indicates that phosphorylated GAP43 is mainly present in the pm fraction. (C) Image of a single focal plane of a living PC12 cell expressing PAGFP-F (top) and immunoblot showing the distribution of PAGFP-F (GFP), tubulin (tub), and transferrin receptor (TFR) in the indicated fractions. Note the high enrichment of PAGFP-F in the pm fraction. Scale bar, 10 μm. Numbers to the sides of the gel blots indicate molecular mass standards in kilodaltons (B, C). Statistical analysis was performed using Student's t test. ***, p

    Article Snippet: The following antibodies were used: anti-GFP (rabbit polyclonal; Millipore, Billerica, MA), anti-phosphoneuromodulin (rabbit antiserum; Upstate, Lake Placid, NY), anti-tubulin (DM1A; mouse monoclonal), and anti-TRF (mouse monoclonal; Invitrogen).

    Techniques: Expressing, Construct, Imaging, Quantitation Assay, Fractionation, Western Blot

    Morphological analysis of MGHs. a Phase-contrast microscopic image of an MGH. b Transmission electron microscopic cross-section of an MGH. The arrows indicate the nuclei. c TRITC-phalloidin staining (red) of hemocytes from immune-induced D. ananassae larvae. d Anti-tubulin (green) and nuclear DAPI staining (blue) of the untreated and colchicine-treated MGHs of immune-induced larvae. Scale bars = 5 ( b ), 25 ( d ) and 50 μm ( a , c ; see online version for colors).

    Journal: Journal of Innate Immunity

    Article Title: Multinucleated Giant Hemocytes Are Effector Cells in Cell-Mediated Immune Responses of Drosophila

    doi: 10.1159/000369618

    Figure Lengend Snippet: Morphological analysis of MGHs. a Phase-contrast microscopic image of an MGH. b Transmission electron microscopic cross-section of an MGH. The arrows indicate the nuclei. c TRITC-phalloidin staining (red) of hemocytes from immune-induced D. ananassae larvae. d Anti-tubulin (green) and nuclear DAPI staining (blue) of the untreated and colchicine-treated MGHs of immune-induced larvae. Scale bars = 5 ( b ), 25 ( d ) and 50 μm ( a , c ; see online version for colors).

    Article Snippet: Phalloidin-rhodamine, anti-tubulin (mouse monoclonal antibody, 1:30 dilution), anti-bromodeoxyuridine (BrdU; Alexa Fluor 488-conjugated mouse monoclonal antibody, 1:10 dilution) and anti-GFP (rabbit polyclonal antibody, 1:2,000 dilution) were from Invitrogen; anti-phospho-histone H3 (rabbit polyclonal antibody, 1:10,000 dilution) was from Sigma.

    Techniques: Transmission Assay, Staining

    Silencing of integrin β1 inhibits IGFBP3-induced migration A. The activities of Cdc42, Rac1 and RhoA were detected in IGBBP3 knockdown cells (IGFBP3 sh4 and sh5) and the corresponding controls (pLKO-GFP). Equal amounts of input protein were subjected to Western blot using anti-Cdc42 (total Cdc42), anti-Rac1 (total Rac1), and anti-RhoA (total RhoA) antibodies. Equal amounts of protein were incubated with GST-PAK1 (detection of active Cdc42 or Rac1) and GST-Rhotekin (detection of active RhoA). Complexes were collected with gluthathione-Sepharose and resolved by Western blot. GTPγS was served as a positive control. Anti-GST antibodies were served as a loading control. B. The density of each band was measured by image J and normalized with the controls (LN1–1 pLKO-GFP). The activities of active small GTPase were conducted by dividing the density of active small GTPase to that of GST loading controls. The relative activities were obtained when the activity of active small GTPase in LN1–1 pLKO-GFP were set to 1. C. Representative data shows the relative migration activity of OEC-M1 cells with dominant-negative Cdc42 (Cdc42dn) and the corresponding controls (PB). The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 PB treated with treated with IGFBP3 and PB-Cdc42dn cells with/without IGFBP3 treatment with that in OEC-M1 PB cells. D. Representative data shows the relative migration activity of IGFBP3 expressing cells (OEC-M1 IGFBP3) and the corresponding controls (OEC-M1 PB) with anti-integrin β1 (200 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 PB and IGFBP3 cells treated with anti-integrin β1 or OEC-M1 IGFBP3 cells treated with IgG antibodies (200 ng/ml) to that in OEC-M1 PB cells treated with IgG antibodies. E. Immunoblot analysis of integrin β1 protein in OEC-M1 cells with ITGB1 shRNA expression (OEC-M1 ITGB1 sh3 and sh4) and vector controls (OEC-M1 pLKO-GFP). α-tubulin serves as an internal control. F. Representative data shows the relative migration activity of OEC-M1 pLKO-GFP, ITGB1 sh3 and sh4 cells upon IGFBP3 (100 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 with ITGB1 knockdown and IGFBP3 treatment with that in the control cells. G. Representative data showed the relative migration activity of OEC-M1 treated with 10, 20 uM of PD98059 (PD) and dimethyl sulfoxide (DMSO) upon IGFBP3 (100 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell/per field in OEC-M1 treated with PD98059 and IGFBP3 with that in the control cells. H. Immunoblot analysis revealed knockdown of ITGB1 inhibited IGFBP3-induced ERK phosphorylation at different time points in OEC-M1 cells (upper panel, 0, untreated; 10, 10 min; 30, 30 min for 100 ng/ml IGFBP3 treatment). The ratio of phosphorylated ERK/total ERK was obtained by dividing the intensity of phosphorylated ERK to that of total ERK. The relative expression was obtained when the ration of untreated cells were set to 1 (lower panel). Bar, SE; ** p

    Journal: Oncotarget

    Article Title: Insulin-like growth factor-independent insulin-like growth factor binding protein 3 promotes cell migration and lymph node metastasis of oral squamous cell carcinoma cells by requirement of integrin β1

    doi:

    Figure Lengend Snippet: Silencing of integrin β1 inhibits IGFBP3-induced migration A. The activities of Cdc42, Rac1 and RhoA were detected in IGBBP3 knockdown cells (IGFBP3 sh4 and sh5) and the corresponding controls (pLKO-GFP). Equal amounts of input protein were subjected to Western blot using anti-Cdc42 (total Cdc42), anti-Rac1 (total Rac1), and anti-RhoA (total RhoA) antibodies. Equal amounts of protein were incubated with GST-PAK1 (detection of active Cdc42 or Rac1) and GST-Rhotekin (detection of active RhoA). Complexes were collected with gluthathione-Sepharose and resolved by Western blot. GTPγS was served as a positive control. Anti-GST antibodies were served as a loading control. B. The density of each band was measured by image J and normalized with the controls (LN1–1 pLKO-GFP). The activities of active small GTPase were conducted by dividing the density of active small GTPase to that of GST loading controls. The relative activities were obtained when the activity of active small GTPase in LN1–1 pLKO-GFP were set to 1. C. Representative data shows the relative migration activity of OEC-M1 cells with dominant-negative Cdc42 (Cdc42dn) and the corresponding controls (PB). The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 PB treated with treated with IGFBP3 and PB-Cdc42dn cells with/without IGFBP3 treatment with that in OEC-M1 PB cells. D. Representative data shows the relative migration activity of IGFBP3 expressing cells (OEC-M1 IGFBP3) and the corresponding controls (OEC-M1 PB) with anti-integrin β1 (200 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 PB and IGFBP3 cells treated with anti-integrin β1 or OEC-M1 IGFBP3 cells treated with IgG antibodies (200 ng/ml) to that in OEC-M1 PB cells treated with IgG antibodies. E. Immunoblot analysis of integrin β1 protein in OEC-M1 cells with ITGB1 shRNA expression (OEC-M1 ITGB1 sh3 and sh4) and vector controls (OEC-M1 pLKO-GFP). α-tubulin serves as an internal control. F. Representative data shows the relative migration activity of OEC-M1 pLKO-GFP, ITGB1 sh3 and sh4 cells upon IGFBP3 (100 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 with ITGB1 knockdown and IGFBP3 treatment with that in the control cells. G. Representative data showed the relative migration activity of OEC-M1 treated with 10, 20 uM of PD98059 (PD) and dimethyl sulfoxide (DMSO) upon IGFBP3 (100 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell/per field in OEC-M1 treated with PD98059 and IGFBP3 with that in the control cells. H. Immunoblot analysis revealed knockdown of ITGB1 inhibited IGFBP3-induced ERK phosphorylation at different time points in OEC-M1 cells (upper panel, 0, untreated; 10, 10 min; 30, 30 min for 100 ng/ml IGFBP3 treatment). The ratio of phosphorylated ERK/total ERK was obtained by dividing the intensity of phosphorylated ERK to that of total ERK. The relative expression was obtained when the ration of untreated cells were set to 1 (lower panel). Bar, SE; ** p

    Article Snippet: Primary antibodies were used as follows: anti-IGFBP3 (MAB305, R & D Systems), anti-integrin β1 (Santa Cruz, Dallas, TX, USA), anti-α-tubulin (MS-581-P0, Thermo Scientific), anti-focal adhesion kinase (FAK, sc-557, Santa Cruz), anti-phosphorylated FAK (611806, BD), anti-Src (#2109, Cell Signaling, Beverly, MA, USA), anti-phosphorylated Src (05–677, Millipore), anti-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB, sc-8008, Santa Cruz), anti-phosphorylated NF-kB (#3033, Cell Signaling), anti-protein kinase B/AKT (#9272, Cell Signaling), anti-phosphorylated AKT (#9271, Cell Signaling), anti-ERK (sc-94, Santa Cruz). anti-phospho-ERK (sc-7383, Santa Cruz), anti-integrin-linked kinase (ILK, GTX101691, GeneTex, Irvine, CA, USA), MMP1 (RB1536P0, Thermo Scientific) and MMP10 (AP6194a, Abgent, San Diego, CA, USA).

    Techniques: Migration, Western Blot, Incubation, Positive Control, Activity Assay, Dominant Negative Mutation, Expressing, shRNA, Plasmid Preparation

    IGF-independent IGFBP3 increases migration and transendothelial migration A. Representative data shows the relative activities of migration and B. transendothelial migration of LN1–1 cells with IGFBP3 knockdown (IGFBP3 sh4 and sh5) and the corresponding controls (pLKO-GFP). The relative migration/transmigration activity was defined by normalizing the mean of migrated cell/per field in LN1–1 IGFBP3 sh4 or sh5 with that in LN1–1 pLKO-GFP cells. C. Immunoblot analysis of IGFBP3 protein in OEC-M1 cells with ectopic wild type and mutant IGFBP3 expression (OEC-M1 IGFBP3, GGG) and vector controls (OEC-M1 PB). α-tubulin serves as an internal control. D. Representative data shows the relative activities of migration and E. transendothelial migration of OEC-M1 IGFBP3, GGG and OEC-M1 PB cells. The relative migration/transmigration activity was defined by normalizing the mean of migrated cells/per field in OEC-M1 IGFBP3 or GGG cells with that in OEC-M1 PB cells. F. Representative data shows the relative migration activity of OEC-M1 cells treated with recombinant IGFBP3. G. Representative data shows the relative transmigration activity of OEC-M1 IGFBP3, OEC-M1 PB cells and OEC-M1 PB cells treated with IGFBP3 proteins. The relative migration/transendothelial migration activity was defined by normalizing the mean of migrated cells/per field in OEC-M1 IGFBP3 or IGFBP3 treated cells with that in the control cells. Bar, SE; * p

    Journal: Oncotarget

    Article Title: Insulin-like growth factor-independent insulin-like growth factor binding protein 3 promotes cell migration and lymph node metastasis of oral squamous cell carcinoma cells by requirement of integrin β1

    doi:

    Figure Lengend Snippet: IGF-independent IGFBP3 increases migration and transendothelial migration A. Representative data shows the relative activities of migration and B. transendothelial migration of LN1–1 cells with IGFBP3 knockdown (IGFBP3 sh4 and sh5) and the corresponding controls (pLKO-GFP). The relative migration/transmigration activity was defined by normalizing the mean of migrated cell/per field in LN1–1 IGFBP3 sh4 or sh5 with that in LN1–1 pLKO-GFP cells. C. Immunoblot analysis of IGFBP3 protein in OEC-M1 cells with ectopic wild type and mutant IGFBP3 expression (OEC-M1 IGFBP3, GGG) and vector controls (OEC-M1 PB). α-tubulin serves as an internal control. D. Representative data shows the relative activities of migration and E. transendothelial migration of OEC-M1 IGFBP3, GGG and OEC-M1 PB cells. The relative migration/transmigration activity was defined by normalizing the mean of migrated cells/per field in OEC-M1 IGFBP3 or GGG cells with that in OEC-M1 PB cells. F. Representative data shows the relative migration activity of OEC-M1 cells treated with recombinant IGFBP3. G. Representative data shows the relative transmigration activity of OEC-M1 IGFBP3, OEC-M1 PB cells and OEC-M1 PB cells treated with IGFBP3 proteins. The relative migration/transendothelial migration activity was defined by normalizing the mean of migrated cells/per field in OEC-M1 IGFBP3 or IGFBP3 treated cells with that in the control cells. Bar, SE; * p

    Article Snippet: Primary antibodies were used as follows: anti-IGFBP3 (MAB305, R & D Systems), anti-integrin β1 (Santa Cruz, Dallas, TX, USA), anti-α-tubulin (MS-581-P0, Thermo Scientific), anti-focal adhesion kinase (FAK, sc-557, Santa Cruz), anti-phosphorylated FAK (611806, BD), anti-Src (#2109, Cell Signaling, Beverly, MA, USA), anti-phosphorylated Src (05–677, Millipore), anti-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB, sc-8008, Santa Cruz), anti-phosphorylated NF-kB (#3033, Cell Signaling), anti-protein kinase B/AKT (#9272, Cell Signaling), anti-phosphorylated AKT (#9271, Cell Signaling), anti-ERK (sc-94, Santa Cruz). anti-phospho-ERK (sc-7383, Santa Cruz), anti-integrin-linked kinase (ILK, GTX101691, GeneTex, Irvine, CA, USA), MMP1 (RB1536P0, Thermo Scientific) and MMP10 (AP6194a, Abgent, San Diego, CA, USA).

    Techniques: Migration, Transmigration Assay, Activity Assay, Mutagenesis, Expressing, Plasmid Preparation, Recombinant