anti actin antibody  (Millipore)

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Fibroblast Growth Factor-9 Activates c-Kit Progenitor Cells and Enhances Angiogenesis in the Infarcted Diabetic Heart

doi: 10.1155/2016/5810908

Figure Lengend Snippet: FGF-9 enhances c-Kit activation and vascular smooth muscle cell differentiation in post-MI C57BL/6 and db/db mice. Representative photomicrographs in (a) depict c-Kit +ve progenitor cells in red (A, E, I, M, Q, and U), SM α -actin VSM cells in green (B, F, J, N, R, and V), total nuclei in blue (C, G, K, O, S, and W), and merged images (D, H, L, P, T, and X) for C57BL/6 and db/db groups. Enlarged images depicting colocalization of c-Kit +ve and SM α -actin +ve cells are presented in yellow boxes in (a) (D, H, L, P, T, and X). Scale bar = 100 μ m. (b) The number of c-Kit +ve /SM α -actin +ve cells is significantly enhanced in the MI + FGF-9 group compared to the MI group in both C57BL/6 and db/db infarcted hearts. ∗ p

Article Snippet: After TUNEL staining, heart sections were costained overnight with primary antibodies against anti-SM α -actin (vascular smooth muscle cells, 1 : 100, cat # A2172-0.2 mL, Sigma Aldrich) followed by an antimouse antibody (M.O.M. kit, cat # FMK-2201, Vector laboratories).

Techniques: Activation Assay, Cell Differentiation, Mouse Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Fibroblast Growth Factor-9 Activates c-Kit Progenitor Cells and Enhances Angiogenesis in the Infarcted Diabetic Heart

doi: 10.1155/2016/5810908

Figure Lengend Snippet: FGF-9 blunts vessel apoptosis in the diabetic infarcted heart. Representative photomicrographs illustrating vascular apoptosis are depicted in (a) for C57BL/6 (A–L) and db/db (M–X) mice with TUNEL positive nuclei in red (A, E, I, M, Q, and U), SM α -actin +ve cells in green (B, F, J, N, R, and V), total nuclei stained blue with DAPI (C, G, K, O, S, and W), and merged images (D, H, L, P, T, and X). The boxes on the top right panel of (a) are enhanced images from each group to demonstrate the colocalization of TUNEL, SM α -actin, and DAPI within a single vessel. Scale bar = 100 μ m. (b) Histogram of quantitative vessels apoptosis in infarcted C57BL/6 and db/db mice. # p

Article Snippet: After TUNEL staining, heart sections were costained overnight with primary antibodies against anti-SM α -actin (vascular smooth muscle cells, 1 : 100, cat # A2172-0.2 mL, Sigma Aldrich) followed by an antimouse antibody (M.O.M. kit, cat # FMK-2201, Vector laboratories).

Techniques: Mouse Assay, TUNEL Assay, Staining

Journal: The EMBO Journal

Article Title: CALCOCO1 acts with VAMP‐associated proteins to mediate ER‐phagy

doi: 10.15252/embj.2019103649

Figure Lengend Snippet: CALCOCO 1 promotes basal autophagic flux but not bulk autophagy HeLa CALCOCO1 KO cells stably expressing EGFP‐CALCOCO1 were starved for 4 h and then immunostained with anti‐ATG13 and anti‐WIPI2 antibodies. Scale bars in (A) are 5 μm for the confocal microscopy images and 2 μm for the airyscans. In (B), the error bars represent mean ± SD of puncta per cell from three independent experiments and 100–200 cells per each experiment. Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH or actin). The asterisk in (K) indicates that endogenous CALCOCO1 is detected in WT and KO cell extracts and EGFP‐CALCOCO1 in cells extracts from the rescued cells. In (D, F, H, I, and L), the bars represent the mean ± SD of band intensities relative to the actin or GAPDH loading control as quantified using ImageJ, n = 5 in (I), n = 3 in others. Statistical comparison was analyzed by one‐way ANOVA and significance displayed as *** P ˂ 0.001, ** P ˂ 0.005, * P ˂ 0.01; ns is not significant.

Article Snippet: AntibodiesMouse monoclonal anti‐CALCOCO1 (A‐10) (Santa Cruz Biotech Cat#sc‐515670), rabbit polyclonal anti‐CALCOCO1 (Sigma‐Aldrich Cat#HPA038314), mouse polyclonal anti‐CALCOCO1 (Abcam Cat# ab167237), rabbit polyclonal anti‐VAPA (Proteintech Cat#15275‐1‐AP), rabbit polyclonal anti‐VAPB (clone 4F6A6) (Proteintech Cat#66191‐1‐IG), rabbit polyclonal anti‐GFP (Abcam Cat #ab290), mouse monoclonal anti‐p62 (BD Biosciences Cat #610833), guinea pig polyclonal anti‐p62 (Progen Cat #GP62‐C), rabbit polyclonal anti‐CALCOCO2 (Sigma‐Aldrich Cat #HPA023195), rabbit anti‐CALCOCO2 (Abcam Cat #ab68588), rabbit monoclonal anti‐ATG7 (Cell Signaling Cat #D12B11), rabbit polyclonal anti‐LC3B (Novus Bio Cat #NB100‐2220), rabbit polyclonal anti‐LC3B (Sigma‐Aldrich Cat # L7543), mouse monoclonal anti‐GABARAP (MBL Cat # M135‐3), mouse monoclonal anti‐Myc tag (9B11) cell signaling #2276), mouse monoclonal anti‐RTN3 (F‐6) (Santa Cruz Biotechnology Cat #sc‐374599), rabbit polyclonal FAM134B (Proteintech Cat #21537‐1‐AP), mouse polyclonal anti‐NBR1 (Santa cruz biotechnology #sc‐130380), rabbit polyclonal anti‐CKAP4(CLIMP63) (Proteintech Cat#16686‐1‐AP), rabbit polyclonal anti‐TEX264 (Novus Bio Cat #NBP1‐89866), rabbit polyclonal anti‐GAPDH (Sigma‐Aldrich Cat#G9545), rabbit polyclonal anti‐actin (Sigma‐Aldrich Cat #A2066), HRP‐conjugated goat polyclonal anti‐rabbit (BD Biosciences Cat #554021), HRP‐conjugated goat polyclonal anti‐mouse (BD Biosciences Cat #554002).

Techniques: Stable Transfection, Expressing, Confocal Microscopy

Journal: Infection and Immunity

Article Title: Role of Src Kinases in Mobilization of Glycosylphosphatidylinositol-Anchored Decay-Accelerating Factor by Dr Fimbria-Positive Adhering Bacteria ▿Role of Src Kinases in Mobilization of Glycosylphosphatidylinositol-Anchored Decay-Accelerating Factor by Dr Fimbria-Positive Adhering Bacteria ▿ †

doi: 10.1128/IAI.01052-10

Figure Lengend Snippet: The CCP4 domain of hDAF is essential for Src kinase activation and hDAF mobilization around adhering Dr-positive E. coli . (A) CHO-hDAF cells and CHO cells stably expressing hDAF deleted of its CCP1 or CCP4 domain (CHO-hDAFΔCCP1 and CHO-hDAFΔCCP4) were infected with Dr-positive E. coli (Dr+). The mobilization of hDAF, of mutated hDAFΔCCP1, or of mutated hDAFΔCCP4 around Dr-positive E. coli was observed by indirect immunofluorescence, followed by a 3D reconstruction of a cross-section of images (white square). The AF2009 and J63 antibodies were used to detect hDAF (red) and the Dr fimbriae (green), respectively. (B) CHO-hDAF, CHO-hDAFΔCCP1, or CHO-hDAFΔCCP4 cells were infected with IH11128 strain (wt) or Dr-positive E. coli or were not infected (NI). Proteins were analyzed in SDS-PAGE and Western immunoblotted with appropriate antibodies to detect activated Src kinases (Src-Y416 P ), Src kinase protein (Src), and actin (top panel) and to quantify them (lower panel). Micrographs are representative of three independent experiments. Immunoblots are representative of three independent experiments. Blotting with antiactin pAb was used to confirm gel loading. Blots were quantified using Image J software. The values represent the mean ± SEM. *, P

Article Snippet: The antibodies used were goat anti-hDAF polyclonal antibody (pAb) (AF2009) from R & D systems; mouse monoclonal antibody (MAb) 8D11, directed against the CCP4 domain of hDAF (Department of Pathology, Washington University School of Medicine, St. Louis, MO); rabbit anti-phospho-Src Tyr416 pAb, which recognizes phospho-Tyr416 (mammals) or phospho-Tyr418 (human), and rabbit anti-Src pAb, purchased from Cell Signaling (Denvers, MA); mouse anti-c-Src MAb (Millipore); mouse anti-Yes MAb (BD Transduction Laboratories); mouse anti-Fyn MAb and mouse anti-Lyn MAb (Exbio); rabbit antiactin pAb (Sigma); antiphosphotyrosine pY20 MAb (BD Transduction Laboratories); anti-CEACAM pAb (Dako Cytomation); anti-CEACAM MAb D14HD11 (Genovac); and rabbit anti-Dr pAb (J63), directed against the DraE adhesin subunit of Dr fimbriae ( ).

Techniques: Activation Assay, Stable Transfection, Expressing, Infection, Immunofluorescence, SDS Page, Western Blot, Software

Journal: Infection and Immunity

Article Title: Role of Src Kinases in Mobilization of Glycosylphosphatidylinositol-Anchored Decay-Accelerating Factor by Dr Fimbria-Positive Adhering Bacteria ▿Role of Src Kinases in Mobilization of Glycosylphosphatidylinositol-Anchored Decay-Accelerating Factor by Dr Fimbria-Positive Adhering Bacteria ▿ †

doi: 10.1128/IAI.01052-10

Figure Lengend Snippet: Mobilization of hDAF around adhering Dr-positive E. coli requires activated Src kinases. (A) Immunoblot analysis of hDAF, activated Src kinases (Src-Y418 P ), Src kinases (Src), and actin in the Triton X-100-extracted fraction (E) and the Triton X-100-resistant fraction (R) isolated from HeLa cells infected with IH11128 strain (wt), Dr-positive (Dr+) or Dr-negative (Dr−) E. coli , or not infected (NI). (Left panel) the different fractions were analyzed by SDS-PAGE, followed by Western blotting with appropriate antibodies. (Right panel) Quantification of activated Src kinases (Src-Y418 P ). (B) Knockdown expression of hDAF by specific siRNA in HeLa cells. The HeLa cells were transfected or not transfected (NT) with a control or with a specific hDAF siRNA. hDAF expression was analyzed by Western blotting using anti-hDAF AF2009 antibody (left panel) and quantified (right panel). (C) Knockdown expression of hDAF results in a decrease in Dr-positive E. coli -induced activation of Src kinase. Western blotting (left panel) and quantification (right panel) of activated Src kinases (Src-Y418 P ). (D) The Src kinase inhibitor PP2 inhibited the Dr-positive E. coli -induced activation of Src kinase, whereas its inactive analog, PP3, did not. Western blotting (top panel) and quantification (lower panel) of activated Src kinases (Src-Y418 P ). Each immunoblot (A to D) is representative of three independent experiments. Blotting with antiactin pAb was used to confirm gel loading. Blots were quantified using Image J software. The value reported represents the mean ± SEM. *, P

Article Snippet: The antibodies used were goat anti-hDAF polyclonal antibody (pAb) (AF2009) from R & D systems; mouse monoclonal antibody (MAb) 8D11, directed against the CCP4 domain of hDAF (Department of Pathology, Washington University School of Medicine, St. Louis, MO); rabbit anti-phospho-Src Tyr416 pAb, which recognizes phospho-Tyr416 (mammals) or phospho-Tyr418 (human), and rabbit anti-Src pAb, purchased from Cell Signaling (Denvers, MA); mouse anti-c-Src MAb (Millipore); mouse anti-Yes MAb (BD Transduction Laboratories); mouse anti-Fyn MAb and mouse anti-Lyn MAb (Exbio); rabbit antiactin pAb (Sigma); antiphosphotyrosine pY20 MAb (BD Transduction Laboratories); anti-CEACAM pAb (Dako Cytomation); anti-CEACAM MAb D14HD11 (Genovac); and rabbit anti-Dr pAb (J63), directed against the DraE adhesin subunit of Dr fimbriae ( ).

Techniques: Isolation, Infection, SDS Page, Western Blot, Expressing, Transfection, Activation Assay, Software

Journal: Infection and Immunity

Article Title: Role of Src Kinases in Mobilization of Glycosylphosphatidylinositol-Anchored Decay-Accelerating Factor by Dr Fimbria-Positive Adhering Bacteria ▿Role of Src Kinases in Mobilization of Glycosylphosphatidylinositol-Anchored Decay-Accelerating Factor by Dr Fimbria-Positive Adhering Bacteria ▿ †

doi: 10.1128/IAI.01052-10

Figure Lengend Snippet: Recombinant E. coli expressing the Dr fimbriae mutated at position 54 of DraE adhesin subunit does not activate Src kinases and does not mobilize hDAF. (A) Immunofluorescence analysis of hDAF mobilization around bacteria in HeLa cells infected with bacteria expressing Dr fimbriae (Dr + ) or Dr fimbriae mutated at position 54 (Dr+D54C). The white square indicates the cross-section used for 3D analysis. The AF2009 and J63 antibodies were used to detect hDAF (red) and the Dr fimbriae (green), respectively. (B) HeLa cells were infected with Dr-negative E. coli (Dr−) or E. coli expressing Dr fimbriae (Dr+) or mutated adhesin subunit (Dr+D54C) or were not infected (NI). Proteins were analyzed by SDS-PAGE and Western immunoblotted with appropriate antibodies to detect activated Src kinase (Src-Y418 P ), Src kinase proteins (Src), and actin (top panel) and quantify them (lower panel). Micrographs are representative of three independent experiments. Immunoblots are representative of three independent experiments. Blotting with antiactin pAb was used to confirm gel loading. Blots were quantified using Image J software. The value reported represents the mean ± SEM. *, P

Article Snippet: The antibodies used were goat anti-hDAF polyclonal antibody (pAb) (AF2009) from R & D systems; mouse monoclonal antibody (MAb) 8D11, directed against the CCP4 domain of hDAF (Department of Pathology, Washington University School of Medicine, St. Louis, MO); rabbit anti-phospho-Src Tyr416 pAb, which recognizes phospho-Tyr416 (mammals) or phospho-Tyr418 (human), and rabbit anti-Src pAb, purchased from Cell Signaling (Denvers, MA); mouse anti-c-Src MAb (Millipore); mouse anti-Yes MAb (BD Transduction Laboratories); mouse anti-Fyn MAb and mouse anti-Lyn MAb (Exbio); rabbit antiactin pAb (Sigma); antiphosphotyrosine pY20 MAb (BD Transduction Laboratories); anti-CEACAM pAb (Dako Cytomation); anti-CEACAM MAb D14HD11 (Genovac); and rabbit anti-Dr pAb (J63), directed against the DraE adhesin subunit of Dr fimbriae ( ).

Techniques: Recombinant, Expressing, Immunofluorescence, Infection, SDS Page, Western Blot, Software

Journal: Journal of Virology

Article Title: Studies on the Interaction of Tumor-Derived HD5 Alpha Defensins with Adenoviruses and Implications for Oncolytic Adenovirus Therapy

doi: 10.1128/JVI.02030-16

Figure Lengend Snippet: K562 cells with ectopic HD5 expression. (A) Screening of cell lines for endogenous HD5 expression. HD5 mRNA levels were measured in total cellular RNA by qRT-PCR and expressed as percentages of β-actin (Act B) mRNA levels. (B) HD5 mRNA expression

Article Snippet: After blots were stripped with Restore Western blot striping buffer (Thermo Scientific, Waltham, MA), they were incubated with mouse anti-β-actin (Sigma, St. Louis, MO) (1:5,000).

Techniques: Expressing, Quantitative RT-PCR, Activated Clotting Time Assay