sds-polyacrylamide gel electrophoresis sds-page Search Results


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  • 99
    Thermo Fisher sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page
    LPS-Trap-Fc1 to -Fc4 bind LPS and block LPS-mediated IL-6 production in Mono Mac 6 cells. (A) Supernatants of HEK293T cells transfected with LPS-Trap-Fc1 were incubated in the presence (lane 1) or absence (lane 3) of biotin-LPS. Additionally, supernatants were preincubated with an anti-TLR4/MD-2 MAb (lane 2) or an excess of LPS (lane 4). Complexes were immunoprecipitated with streptavidin-Sepharose, subjected to <t>SDS-PAGE,</t> and analyzed by Western blotting with an anti-FLAG MAb. (B) RAW 264.7 cells were incubated with LPS-Trap-Fc1 or a medium control. Cells were stimulated with 100 ng/ml LPS overnight, and IL-6 levels in supernatants were determined by ELISA. *, P
    Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Sds Page, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6011 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page
    Various expression patterns of VP7 in transfected cells. (A) Purification of VP7 with Ni 2 + column chromatography and detection by <t>SDS-PAGE</t> and Western blotting. Primary antibody was 6*His antibody. M. Protein molecular mass marker; 1–7: The purified protein eluted by 10, 20, 50, 100, 200, 300, 400 mmol/L of imidazole solution respectively. (B) Purification of VP7 detected by Western blotting. Primary antibody was VP7 antibody.
    Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Sds Page, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 18104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page
    Protein analysis of M. tuberculosis. (A) <t>SDS-PAGE</t> analysis of protein extracts from M. tuberculosis stained with Coomassie blue. Cultures were incubated for the indicated times in slowly stirred sealed tubes. AG indicates shaking aerobic incubation for 80 h. The alpha-crystallin homologue is indicated. (B) Immunoblot analysis of the Acr protein. Proteins from either aerobic (AG) or 141-h-old hypoxic cultures were probed with either monoclonal anti-Acr (CS49) or polyclonal anti-URB-1 (RS88-13078) antibody.
    Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Sds Page, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 13151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore sds polyacrylamide gel electrophoresis sds page
    Down regulation of cIAP-1, cIAP-2, XIAP, MDM2 and activation of p53 AGS and SNU-484 were treated with indicated concentrations of scutellarein or 24 h. The cell lysates were subjected to <t>SDS–PAGE</t> and analyzed by immune-blotting. Densitometry analyses of cIAP-1,-2, XIAP, MDM2, p53 and p-p53 proteins expressions were expressed as mean ± SD of three independent experiments. ( ** P
    Sds Polyacrylamide Gel Electrophoresis Sds Page, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4185 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Bio-Rad sds page
    Down regulation of cIAP-1, cIAP-2, XIAP, MDM2 and activation of p53 AGS and SNU-484 were treated with indicated concentrations of scutellarein or 24 h. The cell lysates were subjected to <t>SDS–PAGE</t> and analyzed by immune-blotting. Densitometry analyses of cIAP-1,-2, XIAP, MDM2, p53 and p-p53 proteins expressions were expressed as mean ± SD of three independent experiments. ( ** P
    Sds Page, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 4860 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad sds page electrophoresis
    A) Image of cellular targets of EPO in mouse brain separated by <t>SDS-PAGE</t> in a 12 % gel. Gel was stained by Silver. B)Image of cellular targets of EPO in mouse brain separated by IEF in a 7 cm IPG strip containing nonlinear pH gradient 3–10 followed by two-dimensional gel electrophoresis. Protein detection was by Silver-staining
    Sds Page Electrophoresis, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 2705 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad gradient sds page gels
    p53 oligomerization is decreased in p53 334H/H mouse tissues. A , liver tissue lysates were cross-linked with glutaraldehyde, resolved in <t>SDS-PAGE</t> gel, and immunoblotted. Note p53 antibody specificity demonstrated by lack of immunoreactivity in the p53 −/− sample. Protein standards are in kD. Triplicate lanes of each genotype represent liver samples from 3 separate mice. B, fraction of p53 oligomers (T, tetramer; D, dimer; M, monomer) within each lane of immunoblot (A) quantified by densitometry and compared with the respective wild-type oligomer ( n = 3). C , p53 immunoblot of cross-linked liver lysates obtained from mice 6 h after p53 induction by doxorubicin treatment. Duplicate lanes of each genotype represent liver samples from 2 separate mice. D , p53 binding to the p53 response element (p53RE) of p21 in γ-irradiated (γ-IR) mouse liver. ChIP was performed using nonspecific IgG or anti-p53 antibody. p53RE binding is shown relative to wild-type non-specific IgG samples ( n = 3). E, induction of p21 mRNA in the indicated tissues by γ-IR quantified by RT-PCR ( n = 3). Levels were normalized relative to a housekeeping gene TIF . Bone marrow (BM); small intestine. p53 R334 genotypes: wild-type ( R/R ); heterozygous mutant ( R/H ); homozygous mutant ( H/H ); and p53 null ( −/− ). Values are mean ± SD. Compared to wild-type samples within each group, statistical differences were tested by 1-way ANOVA. * P
    Gradient Sds Page Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 1295 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher sds page
    p53 oligomerization is decreased in p53 334H/H mouse tissues. A , liver tissue lysates were cross-linked with glutaraldehyde, resolved in <t>SDS-PAGE</t> gel, and immunoblotted. Note p53 antibody specificity demonstrated by lack of immunoreactivity in the p53 −/− sample. Protein standards are in kD. Triplicate lanes of each genotype represent liver samples from 3 separate mice. B, fraction of p53 oligomers (T, tetramer; D, dimer; M, monomer) within each lane of immunoblot (A) quantified by densitometry and compared with the respective wild-type oligomer ( n = 3). C , p53 immunoblot of cross-linked liver lysates obtained from mice 6 h after p53 induction by doxorubicin treatment. Duplicate lanes of each genotype represent liver samples from 2 separate mice. D , p53 binding to the p53 response element (p53RE) of p21 in γ-irradiated (γ-IR) mouse liver. ChIP was performed using nonspecific IgG or anti-p53 antibody. p53RE binding is shown relative to wild-type non-specific IgG samples ( n = 3). E, induction of p21 mRNA in the indicated tissues by γ-IR quantified by RT-PCR ( n = 3). Levels were normalized relative to a housekeeping gene TIF . Bone marrow (BM); small intestine. p53 R334 genotypes: wild-type ( R/R ); heterozygous mutant ( R/H ); homozygous mutant ( H/H ); and p53 null ( −/− ). Values are mean ± SD. Compared to wild-type samples within each group, statistical differences were tested by 1-way ANOVA. * P
    Sds Page, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 3012 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad sds page gel
    Co-immunoprecipitation of the selective recruitment of Csk and pSHP-2 to various 1.1b WT CYT constructs following co-crosslinking with 2.6b ITAM CYT . AD293 cells (3 × 10 5 ) co-expressing 2.6b ITAM CYT and the various 1.1b CYT constructs were incubated at 37 °C for 8 min with 3 µm magnetic beads (3 × 10 6 ) opsonized with the indicated mAbs and/or isotype IgG1 (’+’ and ‘−’ indicate the presence and absence of corresponding antibodies on beads, respectively). Cells were immediately lysed on ice for 30 min and then magnetic beads were separated, washed three times, and eluted before separating bead-bound proteins using <t>SDS-PAGE.</t> Separated proteins were transferred to nitrocellulose membranes and then probed with α-FLAG and α-HA mAbs ( A ) to verify successful pull-down of the various 1.1b CYT constructs (top membrane) and 2.6b ITAM CYT (bottom membrane). Co-immunoprecipitation of potential effector molecules ( B ) was further examined by probing membranes with α-Csk, α-pSHP-2, α-PTEN, and α-SHIP2 mAbs. For the bottom two panels in ( B ), a whole cell lysate (WCL) sample is included to show that these molecules are present in AD293 lysates. Blots shown are representative examples of three independent experiments.
    Sds Page Gel, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 2115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher bis tris sds page gels
    New monoclonal antibodies visualize parkin in ageing human midbrain. (a-c) Characterization of four murine, monoclonal antibodies (of IgG 2 isotype; clone-B, -E, -D, and -G) in three different assays: (a) against recombinant (r-), full-length, untagged, wildtype (WT) human parkin using non-denaturing slot blots (100ng/slot) of original antigen as well as truncated r-parkin 321-465 and full-length, untagged, human r-DJ-1; (b) against human brain lysates <t>(SDS</t> fractions from control and PRKN- linked ARPD cases) using non-denaturing dot blots; and (c) by denaturing <t>SDS/PAGE</t> and Western blotting of extracts from cortical specimens of a control brain and a parkin-deficient ARPD case. Screening by these three methods as well as by cell-based microscopy (using indirect immunofluorescence) revealed specific staining for four anti-parkin clones (-B, -E, -D and -G), which was conformation-dependent for clone-E. List of epitopes within the sequence of human parkin, as recognized by clones -B, -E, -D, and -G and informed by overlapping screening with 7-12 amino acid-long peptides covering full-length, human parkin. Note that the clone E epitope is conformational, comprised of the three regions indicated.
    Bis Tris Sds Page Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1301 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad sds page loading buffer
    Spy Go from mammalian expression. a HEK293T cells were transfected with the extracellular region of EpCAM fused to SpyTag and a His-tag (EpCAM-SpyTag). EpCAM-SpyTag was purified from the clarified cell supernatant using Spy Go. Fractions were analyzed by <t>SDS-PAGE</t> with Coomassie staining. T: total pooled elutions. Resin: resin post-elution. b Ni-NTA purification of EpCAM-SpyTag as in a . c Spy Go purification of CyRPA-SpyTag from Expi293HEK cells as in a
    Sds Page Loading Buffer, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 1037 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher bis tris sds page gel
    Immunodetection of Cr LPAAT 1 expression in C. reinhardtii cells expressing HA ‐tagged Cr LPAAT 1 using anti‐ HA antibodies. (a) Immunoblot analysis of C. reinhardtii cells expressing HA ‐tagged Cr LPAAT 1 using anti‐ HA antibodies. Two bands were detected; the upper band corresponds to the full protein (36.5 kDa), and the lower band corresponds to the mature protein (31.5 kDa) lacking the transit peptide. (b) The <t>SDS</t> ‐ <t>PAGE</t> gel was stained with blue dye (ProSieve EX Safe Stain— LONZA ) as a loading control for the immunoblot shown in (a). Twenty micrograms of total proteins were loaded onto an SDS ‐ PAGE gel, and expression of Cr LPAAT 1 was detected using anti‐ HA antibodies. A duplicate of the gel was also visualized after staining with blue dye for 1 h. OE : pLM 21‐Cr LPAAT 1‐ HA overexpressors; three transformants ( OE 1, OE 2 and OE 3) are shown.
    Bis Tris Sds Page Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1180 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gradient sds page gels
    KLEIP is a 64-kDa protein and expressed ubiquitously in human adult tissues. (A) Immunoblotting analysis using an affinity-purified anti-KLEIP-N antibody. FLAG-vector (lane 1), FLAG-tagged KLEIP-N (lane 2) or -FL (lane 3) was transfected in COS cells. Cell lysates (30 μg of protein) were separated on 8–16% gradient <t>SDS-PAGE</t> gels and analyzed by the immunoglobulin fraction of preimmune sera (prebleed), purified anti-KLEIP-N antibody or anti-FLAG antibody. Predicted molecular sizes of KLEIP-N and -FL are 64- and 98-kDa, and the locations of these proteins are shown by arrows. Locations of molecular size markers are shown on the left of the panel. (B) Identification of endogenous KLEIP in cultured cells. Lysates of HeLa cells were analyzed by immunoblotting. Prebleed, N and C show immunoblots with the preimmune sera (for anti-KLEIP-N), anti-KLEIP-N or -C, respectively. The location of the predicted KLEIP is shown by an arrow. (C) Immunoprecipitation of KLEIP. HeLa cell lysates (390 μg of protein) were immunoprecipitated by 0.25 μg of the IgG fraction of preimmune sera (lane 1) or anti-KLEIP-N antibody (lane 2), followed by immunoblotting with IgG of preimmune sera or affinity-purified anti-KLEIP-N antibody. An arrow denotes the 64-kDa protein. Asterisks show bands of heavy and light chains of IgG. (D) Identification of endogenous KLEIP in cultured cells. Lysates (10 μg of protein in each) were separated by SDS-PAGE gels and analyzed by immunoblotting with anti-KLEIP-N. Tubulin was detected as a loading control (bottom). (E) mRNA expression of KLEIP in human tissues. PCR was performed to detect expression of KLEIP (top) or glyceraldehyde-3-phosphate dehydrogenase G3PDH (bottom). Lane 1, heart; 2, brain; 3, liver; 4, placenta; 5, lung; 6, kidney; 7, skeletal muscle; 8, pancreas; 9, spleen; 10, thymus; 11, small intestine; 12, testis; 13, ovary; and 14, colon.
    Gradient Sds Page Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 664 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher sds page gel
    <t>SDS-PAGE</t> silver staining of mTHPC-loaded EGa1-conjugated micelles (EGa1-P n micelles) obtained after 10 washes with PBS following the overnight conjugation of micelles with deprotected EGa1-SATA. Native EGa1 was used as a control. The red arrow indicates a band of EGa1 with one polymer chain conjugated.
    Sds Page Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1264 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    LPS-Trap-Fc1 to -Fc4 bind LPS and block LPS-mediated IL-6 production in Mono Mac 6 cells. (A) Supernatants of HEK293T cells transfected with LPS-Trap-Fc1 were incubated in the presence (lane 1) or absence (lane 3) of biotin-LPS. Additionally, supernatants were preincubated with an anti-TLR4/MD-2 MAb (lane 2) or an excess of LPS (lane 4). Complexes were immunoprecipitated with streptavidin-Sepharose, subjected to SDS-PAGE, and analyzed by Western blotting with an anti-FLAG MAb. (B) RAW 264.7 cells were incubated with LPS-Trap-Fc1 or a medium control. Cells were stimulated with 100 ng/ml LPS overnight, and IL-6 levels in supernatants were determined by ELISA. *, P

    Journal: Infection and Immunity

    Article Title: Lipopolysaccharide-Trap-Fc, a Multifunctional Agent To Battle Gram-Negative Bacteria ▿

    doi: 10.1128/IAI.00004-09

    Figure Lengend Snippet: LPS-Trap-Fc1 to -Fc4 bind LPS and block LPS-mediated IL-6 production in Mono Mac 6 cells. (A) Supernatants of HEK293T cells transfected with LPS-Trap-Fc1 were incubated in the presence (lane 1) or absence (lane 3) of biotin-LPS. Additionally, supernatants were preincubated with an anti-TLR4/MD-2 MAb (lane 2) or an excess of LPS (lane 4). Complexes were immunoprecipitated with streptavidin-Sepharose, subjected to SDS-PAGE, and analyzed by Western blotting with an anti-FLAG MAb. (B) RAW 264.7 cells were incubated with LPS-Trap-Fc1 or a medium control. Cells were stimulated with 100 ng/ml LPS overnight, and IL-6 levels in supernatants were determined by ELISA. *, P

    Article Snippet: Samples were separated by 4 to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Nupage Bis-Tris gel; Invitrogen) and electroblotted onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany).

    Techniques: Blocking Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay

    Analysis of LPS-Trap-Fc1 to -Fc4. (A) Schematic representation of the mTLR4/mMD-2 fusion proteins LPS-Trap (left top) and LPS-Trap-Fc1 (left bottom and right). (B) Supernatants of LPS-Trap-Fc1-, -Fc2-, -Fc3-, or -Fc4-transfected HEK293T cells were subjected to SDS-PAGE under reducing conditions and analyzed by Western blotting for the human IgG-Fc tail (left) or the N-terminal FLAG epitope (right).

    Journal: Infection and Immunity

    Article Title: Lipopolysaccharide-Trap-Fc, a Multifunctional Agent To Battle Gram-Negative Bacteria ▿

    doi: 10.1128/IAI.00004-09

    Figure Lengend Snippet: Analysis of LPS-Trap-Fc1 to -Fc4. (A) Schematic representation of the mTLR4/mMD-2 fusion proteins LPS-Trap (left top) and LPS-Trap-Fc1 (left bottom and right). (B) Supernatants of LPS-Trap-Fc1-, -Fc2-, -Fc3-, or -Fc4-transfected HEK293T cells were subjected to SDS-PAGE under reducing conditions and analyzed by Western blotting for the human IgG-Fc tail (left) or the N-terminal FLAG epitope (right).

    Article Snippet: Samples were separated by 4 to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Nupage Bis-Tris gel; Invitrogen) and electroblotted onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany).

    Techniques: Transfection, SDS Page, Western Blot, FLAG-tag

    Various expression patterns of VP7 in transfected cells. (A) Purification of VP7 with Ni 2 + column chromatography and detection by SDS-PAGE and Western blotting. Primary antibody was 6*His antibody. M. Protein molecular mass marker; 1–7: The purified protein eluted by 10, 20, 50, 100, 200, 300, 400 mmol/L of imidazole solution respectively. (B) Purification of VP7 detected by Western blotting. Primary antibody was VP7 antibody.

    Journal: Gene

    Article Title: Identification and characterization of vp7 gene in Bombyx mori cytoplasmic polyhedrosis virus

    doi: 10.1016/j.gene.2017.06.048

    Figure Lengend Snippet: Various expression patterns of VP7 in transfected cells. (A) Purification of VP7 with Ni 2 + column chromatography and detection by SDS-PAGE and Western blotting. Primary antibody was 6*His antibody. M. Protein molecular mass marker; 1–7: The purified protein eluted by 10, 20, 50, 100, 200, 300, 400 mmol/L of imidazole solution respectively. (B) Purification of VP7 detected by Western blotting. Primary antibody was VP7 antibody.

    Article Snippet: The proteins were detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting ( B), using a primary antibody of rabbit anti-6 × His (Sigma) and a secondary antibody of horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Sigma).

    Techniques: Expressing, Transfection, Purification, Column Chromatography, SDS Page, Western Blot, Marker

    Construction of pET-28a-S7 vector and identification of recombinant VP7 and prepared polyclonal VP7 antibody. (A) Electrophoretogram. (B) SDS-PAGE and Western blotting.

    Journal: Gene

    Article Title: Identification and characterization of vp7 gene in Bombyx mori cytoplasmic polyhedrosis virus

    doi: 10.1016/j.gene.2017.06.048

    Figure Lengend Snippet: Construction of pET-28a-S7 vector and identification of recombinant VP7 and prepared polyclonal VP7 antibody. (A) Electrophoretogram. (B) SDS-PAGE and Western blotting.

    Article Snippet: The proteins were detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting ( B), using a primary antibody of rabbit anti-6 × His (Sigma) and a secondary antibody of horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Sigma).

    Techniques: Positron Emission Tomography, Plasmid Preparation, Recombinant, SDS Page, Western Blot

    SsaE, but not SseA, is required for SseB secretion in vitro when SseB is overexpressed. (A) The expression and secretion of SseB in the whole-cell lysate (Cell lysate) and culture supernatant (Supernatant) from the S. enterica serovar Typhimurium wild-type, Δ ssaV , ssaE ( I55G ), and ssaE ( I55G ) harboring pSsaE were analyzed by Western blotting with anti-SseB antibody. (B) The expression and secretion of PipB-2HA in the whole-cell lysate (Cell lysate) and culture supernatant (Supernatant) from the S. enterica serovar Typhimurium wild type, ssaE ( I55G ), Δ ssaL , and Δ ssaLssaE ( I55G ) harboring pPipB-2HA were analyzed by Western blotting with anti-HA antibody. (C) The formation of the SsaE-SseB-SseA ternary complex in the Salmonella cytoplasm was analyzed by FLAG pull-down assays using bacterial lysates from the Salmonella wild-type strain expressing the SseA-2HA from the chromosome and harboring pSsaE-FLAG. Equal amounts of samples (Lysate, Final wash, and Elute) were analyzed by SDS-PAGE and Western blotting with anti-HA (for SseA-2HA), anti-FLAG (for SsaE-FLAG), and anti-SseB antibodies. (D) The S. enterica serovar Typhimurium wild-type strain and isogenic mutants harboring the plasmid pACYC-SseB were grown in LPM medium (pH 5.8), and secreted proteins were precipitated by the addition of tricarboxylic acid. Samples from the secreted protein fraction (Supernatant) and whole-cell lysates (Cell lysate) were analyzed by Western blotting with anti-SseB antibody.

    Journal: Journal of Bacteriology

    Article Title: Functional Characterization of SsaE, a Novel Chaperone Protein of the Type III Secretion System Encoded by Salmonella Pathogenicity Island 2 ▿

    doi: 10.1128/JB.00863-09

    Figure Lengend Snippet: SsaE, but not SseA, is required for SseB secretion in vitro when SseB is overexpressed. (A) The expression and secretion of SseB in the whole-cell lysate (Cell lysate) and culture supernatant (Supernatant) from the S. enterica serovar Typhimurium wild-type, Δ ssaV , ssaE ( I55G ), and ssaE ( I55G ) harboring pSsaE were analyzed by Western blotting with anti-SseB antibody. (B) The expression and secretion of PipB-2HA in the whole-cell lysate (Cell lysate) and culture supernatant (Supernatant) from the S. enterica serovar Typhimurium wild type, ssaE ( I55G ), Δ ssaL , and Δ ssaLssaE ( I55G ) harboring pPipB-2HA were analyzed by Western blotting with anti-HA antibody. (C) The formation of the SsaE-SseB-SseA ternary complex in the Salmonella cytoplasm was analyzed by FLAG pull-down assays using bacterial lysates from the Salmonella wild-type strain expressing the SseA-2HA from the chromosome and harboring pSsaE-FLAG. Equal amounts of samples (Lysate, Final wash, and Elute) were analyzed by SDS-PAGE and Western blotting with anti-HA (for SseA-2HA), anti-FLAG (for SsaE-FLAG), and anti-SseB antibodies. (D) The S. enterica serovar Typhimurium wild-type strain and isogenic mutants harboring the plasmid pACYC-SseB were grown in LPM medium (pH 5.8), and secreted proteins were precipitated by the addition of tricarboxylic acid. Samples from the secreted protein fraction (Supernatant) and whole-cell lysates (Cell lysate) were analyzed by Western blotting with anti-SseB antibody.

    Article Snippet: Samples were run on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride membranes (Immobilon; Millipore).

    Techniques: In Vitro, Expressing, Western Blot, SDS Page, Plasmid Preparation

    SsaE interacts with the SPI-2 T3SS translocator protein SseB. Interaction of SsaE with SseB was analyzed by pull-down assays using bacterial lysates of Salmonella wild-type strains harboring the plasmid pBAP-FLAG as a negative control (A) or pSsaE-FLAG (B). Samples were taken before the binding of beads (Lysate), after the final washing with beads (Final wash), and after the elution of fraction with FLAG peptide (Elute). Equal amounts of samples were analyzed by SDS-PAGE and Western blotting with anti-FLAG, anti-SseB, and anti-PagC antibodies. (C) GST pull-down assay using GST or GST-SseB and probed for SsaE-FLAG. Bacterial lysate of E. coli strain DH5α harboring pSsaE-FLAG was incubated with GST or GST-SseB immobilized glutathione Sepharose beads. Samples from lysates before the addition of beads (Lysate) and proteins retained on beads (Elute) were analyzed by SDS-PAGE and Western blotting with anti-FLAG and anti-GST antibodies. A silver-stained SDS-PAGE gel of the elution fractions from pull-down experiments shows the proteins in each sample (bottom).

    Journal: Journal of Bacteriology

    Article Title: Functional Characterization of SsaE, a Novel Chaperone Protein of the Type III Secretion System Encoded by Salmonella Pathogenicity Island 2 ▿

    doi: 10.1128/JB.00863-09

    Figure Lengend Snippet: SsaE interacts with the SPI-2 T3SS translocator protein SseB. Interaction of SsaE with SseB was analyzed by pull-down assays using bacterial lysates of Salmonella wild-type strains harboring the plasmid pBAP-FLAG as a negative control (A) or pSsaE-FLAG (B). Samples were taken before the binding of beads (Lysate), after the final washing with beads (Final wash), and after the elution of fraction with FLAG peptide (Elute). Equal amounts of samples were analyzed by SDS-PAGE and Western blotting with anti-FLAG, anti-SseB, and anti-PagC antibodies. (C) GST pull-down assay using GST or GST-SseB and probed for SsaE-FLAG. Bacterial lysate of E. coli strain DH5α harboring pSsaE-FLAG was incubated with GST or GST-SseB immobilized glutathione Sepharose beads. Samples from lysates before the addition of beads (Lysate) and proteins retained on beads (Elute) were analyzed by SDS-PAGE and Western blotting with anti-FLAG and anti-GST antibodies. A silver-stained SDS-PAGE gel of the elution fractions from pull-down experiments shows the proteins in each sample (bottom).

    Article Snippet: Samples were run on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride membranes (Immobilon; Millipore).

    Techniques: Plasmid Preparation, Negative Control, Binding Assay, SDS Page, Western Blot, Pull Down Assay, Incubation, Staining

    Immunoblotting assays. A molecular weight marker (Lane a), 20 μg L. infantum SLA (Lane b) and 10 μg rLiHyp (Lanes c, d) were electrophoresed on a 20% SDS-PAGE and blotted onto nitrocellulose membranes. Blots were incubated with pools of sera from B1 phage-vaccinated mice (Lanes b and c) or from naive mice (lane d), and were revealed by adding chloronaphtol, diaminobenzidine, and H 2 O 2 . A scan from the blots is shown here. The black arrow indicates the rLiHyp protein (~28.0 kDa)

    Journal: Parasites & Vectors

    Article Title: Selection strategy of phage-displayed immunogens based on an in vitro evaluation of the Th1 response of PBMCs and their potential use as a vaccine against Leishmania infantum infection

    doi: 10.1186/s13071-017-2576-8

    Figure Lengend Snippet: Immunoblotting assays. A molecular weight marker (Lane a), 20 μg L. infantum SLA (Lane b) and 10 μg rLiHyp (Lanes c, d) were electrophoresed on a 20% SDS-PAGE and blotted onto nitrocellulose membranes. Blots were incubated with pools of sera from B1 phage-vaccinated mice (Lanes b and c) or from naive mice (lane d), and were revealed by adding chloronaphtol, diaminobenzidine, and H 2 O 2 . A scan from the blots is shown here. The black arrow indicates the rLiHyp protein (~28.0 kDa)

    Article Snippet: For this, the recombinant protein and soluble antigenic extract (10 and 20 μg, respectively) were submitted to a 12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and blotted onto a nitrocellulose membrane (0.2 μm pore size, Sigma-Aldrich, St. Louis, Missouri, USA).

    Techniques: Molecular Weight, Marker, SDS Page, Incubation, Mouse Assay

    Protein analysis of M. tuberculosis. (A) SDS-PAGE analysis of protein extracts from M. tuberculosis stained with Coomassie blue. Cultures were incubated for the indicated times in slowly stirred sealed tubes. AG indicates shaking aerobic incubation for 80 h. The alpha-crystallin homologue is indicated. (B) Immunoblot analysis of the Acr protein. Proteins from either aerobic (AG) or 141-h-old hypoxic cultures were probed with either monoclonal anti-Acr (CS49) or polyclonal anti-URB-1 (RS88-13078) antibody.

    Journal: Journal of Bacteriology

    Article Title: Microaerophilic Induction of the Alpha-Crystallin Chaperone Protein Homologue (hspX) mRNA of Mycobacterium tuberculosis

    doi: 10.1128/JB.183.18.5311-5316.2001

    Figure Lengend Snippet: Protein analysis of M. tuberculosis. (A) SDS-PAGE analysis of protein extracts from M. tuberculosis stained with Coomassie blue. Cultures were incubated for the indicated times in slowly stirred sealed tubes. AG indicates shaking aerobic incubation for 80 h. The alpha-crystallin homologue is indicated. (B) Immunoblot analysis of the Acr protein. Proteins from either aerobic (AG) or 141-h-old hypoxic cultures were probed with either monoclonal anti-Acr (CS49) or polyclonal anti-URB-1 (RS88-13078) antibody.

    Article Snippet: Twenty micrograms of protein for each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a Tris-tricine 16.5% polyacrylamide gel (Bio-Rad).

    Techniques: SDS Page, Staining, Incubation

    ) and separated by SDS/PAGE. The glycoproteins and receptors indicated to the Left were identified by WB. For each sample, three replicate gels were developed as follows: one for gB and gD, one for gH and gL, and a third one for αv-integrin and nectin1. ( A ) The complex pulled down by αv STREP β6-integrin in cells transfected with the four glycoproteins, gD, gH, gL, and gB, plus nectin1, αv-integrin, and β6-integrin lacks gL (lane 1, fuchsia star). In contrast, the complex assembled onto αvβ6-integrin alone (lane 4, green star) or nectin1 alone (lane 2) contains gL. ( B ) The complex assembled on αvβ8-integrin in cells transfected with the four glycoproteins, gD, gH, gL, and gB, plus nectin1, αv-integrin, and β8-integrin lacks gL (lane 8, fuchsia star). ( C ) The complex assembled on αvβ6N1-chimera in cells transfected with the four glycoproteins, gD, gH, gL, and gB, plus nectin1, αv-integrin, and β6N-chimera lacks gL (lane 11, fuchsia star). ( D – F ) WB analysis of lysates from samples A – C ). The insoluble material was pelleted. Ten microliters of the supernatant was subjected to SDS/PAGE and WB with antibodies to the indicated proteins. The remaining 90 μL were used for the pulldown experiments shown in A – C .

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Dissociation of HSV gL from gH by αvβ6- or αvβ8-integrin promotes gH activation and virus entry

    doi: 10.1073/pnas.1506846112

    Figure Lengend Snippet: ) and separated by SDS/PAGE. The glycoproteins and receptors indicated to the Left were identified by WB. For each sample, three replicate gels were developed as follows: one for gB and gD, one for gH and gL, and a third one for αv-integrin and nectin1. ( A ) The complex pulled down by αv STREP β6-integrin in cells transfected with the four glycoproteins, gD, gH, gL, and gB, plus nectin1, αv-integrin, and β6-integrin lacks gL (lane 1, fuchsia star). In contrast, the complex assembled onto αvβ6-integrin alone (lane 4, green star) or nectin1 alone (lane 2) contains gL. ( B ) The complex assembled on αvβ8-integrin in cells transfected with the four glycoproteins, gD, gH, gL, and gB, plus nectin1, αv-integrin, and β8-integrin lacks gL (lane 8, fuchsia star). ( C ) The complex assembled on αvβ6N1-chimera in cells transfected with the four glycoproteins, gD, gH, gL, and gB, plus nectin1, αv-integrin, and β6N-chimera lacks gL (lane 11, fuchsia star). ( D – F ) WB analysis of lysates from samples A – C ). The insoluble material was pelleted. Ten microliters of the supernatant was subjected to SDS/PAGE and WB with antibodies to the indicated proteins. The remaining 90 μL were used for the pulldown experiments shown in A – C .

    Article Snippet: Proteins retained by Strep-Tactin Sepharose were separated by SDS polyacrylamide gel electrophoresis (SDS/PAGE), western blotted (WB) with appropriate antibodies, and developed by means of ChemiDoc XRS+ using Image Lab Software (Biorad).

    Techniques: SDS Page, Western Blot, Transfection

    , and separated by SDS/PAGE. Glycoproteins and receptors were identified by WB. It can be seen that the complex assembled onto αvβ6-integrin in the presence of nectin1 lacks gL (lane 3, fuchsia star). When gD (lane 4, HLB), gB (lane 8, DHL), or nectin1 (lanes 5, 6, 9, and αβ6) were missing, the complexes contained gL. ( C and D ) WB analysis of lysates from samples A and B ). The insoluble material was pelleted. Ten microliters of the supernatant was subjected to SDS/PAGE and WB with antibodies to the indicated proteins. The remaining 90 μL were used for the pulldown experiments shown in A and B .

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Dissociation of HSV gL from gH by αvβ6- or αvβ8-integrin promotes gH activation and virus entry

    doi: 10.1073/pnas.1506846112

    Figure Lengend Snippet: , and separated by SDS/PAGE. Glycoproteins and receptors were identified by WB. It can be seen that the complex assembled onto αvβ6-integrin in the presence of nectin1 lacks gL (lane 3, fuchsia star). When gD (lane 4, HLB), gB (lane 8, DHL), or nectin1 (lanes 5, 6, 9, and αβ6) were missing, the complexes contained gL. ( C and D ) WB analysis of lysates from samples A and B ). The insoluble material was pelleted. Ten microliters of the supernatant was subjected to SDS/PAGE and WB with antibodies to the indicated proteins. The remaining 90 μL were used for the pulldown experiments shown in A and B .

    Article Snippet: Proteins retained by Strep-Tactin Sepharose were separated by SDS polyacrylamide gel electrophoresis (SDS/PAGE), western blotted (WB) with appropriate antibodies, and developed by means of ChemiDoc XRS+ using Image Lab Software (Biorad).

    Techniques: SDS Page, Western Blot

    Block of HSV-1 infection by the neutralizing MAb LP11 to gH prevents the dissociation of gL from virion gH/gL. HSV-1(F) virions were preincubated with MAbs 52S, 53S, LP11, or IgGs. J cells expressing αvβ6-integrin plus nectin1 (N+αβ6), or nectin1 alone (N) were exposed to the preincubated virions. After 30 min, the media were harvested and subjected to SDS/PAGE and WB for gL. The medium of cells exposed to LP11-treated virions lacks gL. Lane HSV to the Left shows the WB reactivity of the indicated virion glycoproteins.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Dissociation of HSV gL from gH by αvβ6- or αvβ8-integrin promotes gH activation and virus entry

    doi: 10.1073/pnas.1506846112

    Figure Lengend Snippet: Block of HSV-1 infection by the neutralizing MAb LP11 to gH prevents the dissociation of gL from virion gH/gL. HSV-1(F) virions were preincubated with MAbs 52S, 53S, LP11, or IgGs. J cells expressing αvβ6-integrin plus nectin1 (N+αβ6), or nectin1 alone (N) were exposed to the preincubated virions. After 30 min, the media were harvested and subjected to SDS/PAGE and WB for gL. The medium of cells exposed to LP11-treated virions lacks gL. Lane HSV to the Left shows the WB reactivity of the indicated virion glycoproteins.

    Article Snippet: Proteins retained by Strep-Tactin Sepharose were separated by SDS polyacrylamide gel electrophoresis (SDS/PAGE), western blotted (WB) with appropriate antibodies, and developed by means of ChemiDoc XRS+ using Image Lab Software (Biorad).

    Techniques: Blocking Assay, Infection, Expressing, SDS Page, Western Blot

    SDS-PAGE and Western blot of extracts from parts of the body of adults H. armigera and H. assulta . Upper panels: SDS-PAGE; lower panels: Western blot. (A); antennae, (P): proboscis, (T): tarsi, (W): wings of males (m) and females (f). The expression of OBP7 is limited to antennae with no significant differences between sexes or species. A weak staining in the extract of tarsi might indicate low levels of expression of OBP7 in such organ or cross-reactivity with other OBPs. Molecular weight markers ( M ) are as in Figure 1 .

    Journal: PLoS ONE

    Article Title: A Lysine at the C-Terminus of an Odorant-Binding Protein is Involved in Binding Aldehyde Pheromone Components in Two Helicoverpa Species

    doi: 10.1371/journal.pone.0055132

    Figure Lengend Snippet: SDS-PAGE and Western blot of extracts from parts of the body of adults H. armigera and H. assulta . Upper panels: SDS-PAGE; lower panels: Western blot. (A); antennae, (P): proboscis, (T): tarsi, (W): wings of males (m) and females (f). The expression of OBP7 is limited to antennae with no significant differences between sexes or species. A weak staining in the extract of tarsi might indicate low levels of expression of OBP7 in such organ or cross-reactivity with other OBPs. Molecular weight markers ( M ) are as in Figure 1 .

    Article Snippet: Western Blot Analysis After electrophoretic separation under 14% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), duplicate gels were stained with 0.1% Coomassie blue R250 in 10% acetic acid, 20% ethanol or electroblotted on Trans-Blot nitrocellulose membrane (Bio-Rad Lab) by the procedure of Kyhse-Andersen .

    Techniques: SDS Page, Western Blot, Expressing, Staining, Molecular Weight

    Protein profile of AF S2 and AF S3. (A , B) Representative SDS-PAGE of total soluble leaf proteins (Ext) and apoplastic fluid (AF). (A) Lanes 1 and 3, proteins from 8.64 cm 2 of leaf; lanes 2 and 4, proteins from 0.196 cm 2 of leaf. Arrowheads mark bands in AF that differ between S2 and S3. (B) Each lane contains ~20 μg of protein. MW: molecular weight markers (masses in kDa). Red rectangles show the bands excised and analyzed by mass spectrometry. (C) Densitometric quantification of lanes 1 and 3 from gel A. (D , E) Total protein and peptide content in AF S2 and AF S3, expressed as µg of proteins and peptides per unit of leaf area (D) and as µg of proteins and peptides per gram of leaf fresh weight (LFW), respectively. Mean values ± SEM of biological samples, n. S2 n = 28, S3 n = 30. For each biological sample two technical replicates were taken. Significant differences were calculated at P ≤ 0.05 between leaf stages using the Student's t-test: * , P

    Journal: Frontiers in Plant Science

    Article Title: Physiological and Proteomic Changes in the Apoplast Accompany Leaf Senescence in Arabidopsis

    doi: 10.3389/fpls.2019.01635

    Figure Lengend Snippet: Protein profile of AF S2 and AF S3. (A , B) Representative SDS-PAGE of total soluble leaf proteins (Ext) and apoplastic fluid (AF). (A) Lanes 1 and 3, proteins from 8.64 cm 2 of leaf; lanes 2 and 4, proteins from 0.196 cm 2 of leaf. Arrowheads mark bands in AF that differ between S2 and S3. (B) Each lane contains ~20 μg of protein. MW: molecular weight markers (masses in kDa). Red rectangles show the bands excised and analyzed by mass spectrometry. (C) Densitometric quantification of lanes 1 and 3 from gel A. (D , E) Total protein and peptide content in AF S2 and AF S3, expressed as µg of proteins and peptides per unit of leaf area (D) and as µg of proteins and peptides per gram of leaf fresh weight (LFW), respectively. Mean values ± SEM of biological samples, n. S2 n = 28, S3 n = 30. For each biological sample two technical replicates were taken. Significant differences were calculated at P ≤ 0.05 between leaf stages using the Student's t-test: * , P

    Article Snippet: SDS-PAGE gels (12% w/v acrylamide) were run in a Mini-PROTEAN® III cell (Bio-Rad), and stained with Coomassie-Brilliant Blue R-250 (CBB) or with Coomassie Blue Colloidal G-250 (G-250).

    Techniques: SDS Page, Molecular Weight, Mass Spectrometry

    Dark induced senescence of S2 leaves. (A) Attached S2 leaves (AS2) were wrapped in aluminum foil, and detached S2 leaves (DS2) were placed on moist filter paper in dark boxes, until their chlorophyll content reached S3 values ( Figure 1 ). (B) Leaf chlorophyll content was measured non-destructively with the SPAD meter in different leaves, each of them represents a biological sample (n). S2: n = 29, AS2: n = 22, DS2: n = 5, S3: n = 18. For each biological sample, two SPAD readings were taken. Asterisks represent statistical differences between S2 vs. AS2, DS2 and S3 SPAD values (Dunnett's multiple comparisons test). (C) SDS-PAGE of AF from S2, dark induced attached and detached S2 (AS2 and DS2, respectively) and S3 leaves, compared per leaf area. Arrowheads show AF S3- associated bands.

    Journal: Frontiers in Plant Science

    Article Title: Physiological and Proteomic Changes in the Apoplast Accompany Leaf Senescence in Arabidopsis

    doi: 10.3389/fpls.2019.01635

    Figure Lengend Snippet: Dark induced senescence of S2 leaves. (A) Attached S2 leaves (AS2) were wrapped in aluminum foil, and detached S2 leaves (DS2) were placed on moist filter paper in dark boxes, until their chlorophyll content reached S3 values ( Figure 1 ). (B) Leaf chlorophyll content was measured non-destructively with the SPAD meter in different leaves, each of them represents a biological sample (n). S2: n = 29, AS2: n = 22, DS2: n = 5, S3: n = 18. For each biological sample, two SPAD readings were taken. Asterisks represent statistical differences between S2 vs. AS2, DS2 and S3 SPAD values (Dunnett's multiple comparisons test). (C) SDS-PAGE of AF from S2, dark induced attached and detached S2 (AS2 and DS2, respectively) and S3 leaves, compared per leaf area. Arrowheads show AF S3- associated bands.

    Article Snippet: SDS-PAGE gels (12% w/v acrylamide) were run in a Mini-PROTEAN® III cell (Bio-Rad), and stained with Coomassie-Brilliant Blue R-250 (CBB) or with Coomassie Blue Colloidal G-250 (G-250).

    Techniques: SDS Page

    Physiological characteristics of S1, S2, S3, and S4 leaves. (A) Leaf stages, marked with red arrows, DAE: Days after emergency. (B) Mature, non-senescent leaves (S1 and S2) and senescent leaves (S3 and S4), scale bar 2 cm. (C) Leaf protein content: Representative SDS-PAGE showing a total protein profile loaded per unit leaf area, and immunodetection of Rubisco Large Subunit (RLS), in the left upper and lower panel, respectively. Relative leaf protein content, considering S1 as maximum, was estimated by densitometric analysis of the SDS-PAGE, and it is shown on the right panel. (D) Chlorophyll content per unit leaf area expressed in SPAD units. (E) Maximum quantum yield of Photosystem II (Fv/Fm). (F) Enzymatic activity (U) of the cytosolic enzyme G6PDH in apoplastic fluid (AF) and total leaf extracts (Ext) expressed on the basis of total protein (prot) content. (G) Percentage of G6PDH activity in AF as a function of G6PDH activity in Ext. Values in the graphs show the average ± standard error, SEM, of independent measurements. Data were normally distributed. SPAD readings were taken in 10 to 15 different leaves for each stage, two readings per leaf blade. Fv/Fm values are the average of 16 leaves (S1), 30 leaves (S2 and S3), and six different S4 leaves, one reading per leaf blade. G6PDH activity (D and E) represents the average of biological samples (n), where each FA and Ext biological sample was made up of at least 4 leaves. S1: n = 6 FA and Ext n = 6, S2: n = 4 FA and n = 3 Ext, S3: n = 9 FA and n = 5 Ext, S4: n = 3 FA and 3 Ext. Significant differences were calculated at P ≤ 0.05 between the conditions using the Tukey test; letters indicate significant differences between stages. Asterisks indicate significant differences between AF and Ext; ****, P

    Journal: Frontiers in Plant Science

    Article Title: Physiological and Proteomic Changes in the Apoplast Accompany Leaf Senescence in Arabidopsis

    doi: 10.3389/fpls.2019.01635

    Figure Lengend Snippet: Physiological characteristics of S1, S2, S3, and S4 leaves. (A) Leaf stages, marked with red arrows, DAE: Days after emergency. (B) Mature, non-senescent leaves (S1 and S2) and senescent leaves (S3 and S4), scale bar 2 cm. (C) Leaf protein content: Representative SDS-PAGE showing a total protein profile loaded per unit leaf area, and immunodetection of Rubisco Large Subunit (RLS), in the left upper and lower panel, respectively. Relative leaf protein content, considering S1 as maximum, was estimated by densitometric analysis of the SDS-PAGE, and it is shown on the right panel. (D) Chlorophyll content per unit leaf area expressed in SPAD units. (E) Maximum quantum yield of Photosystem II (Fv/Fm). (F) Enzymatic activity (U) of the cytosolic enzyme G6PDH in apoplastic fluid (AF) and total leaf extracts (Ext) expressed on the basis of total protein (prot) content. (G) Percentage of G6PDH activity in AF as a function of G6PDH activity in Ext. Values in the graphs show the average ± standard error, SEM, of independent measurements. Data were normally distributed. SPAD readings were taken in 10 to 15 different leaves for each stage, two readings per leaf blade. Fv/Fm values are the average of 16 leaves (S1), 30 leaves (S2 and S3), and six different S4 leaves, one reading per leaf blade. G6PDH activity (D and E) represents the average of biological samples (n), where each FA and Ext biological sample was made up of at least 4 leaves. S1: n = 6 FA and Ext n = 6, S2: n = 4 FA and n = 3 Ext, S3: n = 9 FA and n = 5 Ext, S4: n = 3 FA and 3 Ext. Significant differences were calculated at P ≤ 0.05 between the conditions using the Tukey test; letters indicate significant differences between stages. Asterisks indicate significant differences between AF and Ext; ****, P

    Article Snippet: SDS-PAGE gels (12% w/v acrylamide) were run in a Mini-PROTEAN® III cell (Bio-Rad), and stained with Coomassie-Brilliant Blue R-250 (CBB) or with Coomassie Blue Colloidal G-250 (G-250).

    Techniques: SDS Page, Immunodetection, Activity Assay

    Down regulation of cIAP-1, cIAP-2, XIAP, MDM2 and activation of p53 AGS and SNU-484 were treated with indicated concentrations of scutellarein or 24 h. The cell lysates were subjected to SDS–PAGE and analyzed by immune-blotting. Densitometry analyses of cIAP-1,-2, XIAP, MDM2, p53 and p-p53 proteins expressions were expressed as mean ± SD of three independent experiments. ( ** P

    Journal: Oncotarget

    Article Title: Inhibition of IAP’s and activation of p53 leads to caspase-dependent apoptosis in gastric cancer cells treated with Scutellarein

    doi: 10.18632/oncotarget.23202

    Figure Lengend Snippet: Down regulation of cIAP-1, cIAP-2, XIAP, MDM2 and activation of p53 AGS and SNU-484 were treated with indicated concentrations of scutellarein or 24 h. The cell lysates were subjected to SDS–PAGE and analyzed by immune-blotting. Densitometry analyses of cIAP-1,-2, XIAP, MDM2, p53 and p-p53 proteins expressions were expressed as mean ± SD of three independent experiments. ( ** P

    Article Snippet: Proteins were separated by 8%–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Immunobilon-P, 0.45 mm; Millipore, Billerica, MA, USA) using the TE 77 Semi-Dry Transfer Unit (GE Healthcare Life Sciences, Buckinghamshire, UK).

    Techniques: Activation Assay, SDS Page

    Regulatory effect of Scutellarein on cell cycle progression of AGS and SNU484 cells AGS and SNU-484cells were treated with indicated concentrations of scutellarein for 24 h. ( A – B ) Cell cycle distribution was determined by using Cytomics FC 500 (Beckman Coulter, Brea, CA, USA).The data were analyzed using CXP Software. ( C–D ) Effect of Scutellarein on cell cycle-related proteins (cyclin B1, CDK1 and CDC25C) expression level in A549 cells. Cells were treated with Scutellarein (0, 25, 50 and 100 μM) for 24 h. Cell lysates were subjected to SDS–PAGE and analyzed by Western blotting. Representative blots are shown. Densitometric analyses of the effect of Scutellarein on expression of cell cycle-related proteins level were represented. The data are expressed as the mean ± standard deviation (SD) of at least three independent experiments. ( ∗ P

    Journal: Oncotarget

    Article Title: Inhibition of IAP’s and activation of p53 leads to caspase-dependent apoptosis in gastric cancer cells treated with Scutellarein

    doi: 10.18632/oncotarget.23202

    Figure Lengend Snippet: Regulatory effect of Scutellarein on cell cycle progression of AGS and SNU484 cells AGS and SNU-484cells were treated with indicated concentrations of scutellarein for 24 h. ( A – B ) Cell cycle distribution was determined by using Cytomics FC 500 (Beckman Coulter, Brea, CA, USA).The data were analyzed using CXP Software. ( C–D ) Effect of Scutellarein on cell cycle-related proteins (cyclin B1, CDK1 and CDC25C) expression level in A549 cells. Cells were treated with Scutellarein (0, 25, 50 and 100 μM) for 24 h. Cell lysates were subjected to SDS–PAGE and analyzed by Western blotting. Representative blots are shown. Densitometric analyses of the effect of Scutellarein on expression of cell cycle-related proteins level were represented. The data are expressed as the mean ± standard deviation (SD) of at least three independent experiments. ( ∗ P

    Article Snippet: Proteins were separated by 8%–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Immunobilon-P, 0.45 mm; Millipore, Billerica, MA, USA) using the TE 77 Semi-Dry Transfer Unit (GE Healthcare Life Sciences, Buckinghamshire, UK).

    Techniques: Software, Expressing, SDS Page, Western Blot, Standard Deviation

    Caspases activation and subsequent cleavage of PARP in scutellarein -treated AGS and SNU-484cells AGS and SNU-484 cells were treated with indicated concentrations of scutellarein for 24 h. The cell lysates were subjected to SDS–PAGE and analyzed by immune-blotting. Densitometry analyses of Bax/Bcl-xL ratio, Cl.caspase-9, Cl.caspase-3 and Cl.PARP proteins expressions were expressed as the mean ± standard deviation (SD) of at least three independent experiments. ( ∗∗ P

    Journal: Oncotarget

    Article Title: Inhibition of IAP’s and activation of p53 leads to caspase-dependent apoptosis in gastric cancer cells treated with Scutellarein

    doi: 10.18632/oncotarget.23202

    Figure Lengend Snippet: Caspases activation and subsequent cleavage of PARP in scutellarein -treated AGS and SNU-484cells AGS and SNU-484 cells were treated with indicated concentrations of scutellarein for 24 h. The cell lysates were subjected to SDS–PAGE and analyzed by immune-blotting. Densitometry analyses of Bax/Bcl-xL ratio, Cl.caspase-9, Cl.caspase-3 and Cl.PARP proteins expressions were expressed as the mean ± standard deviation (SD) of at least three independent experiments. ( ∗∗ P

    Article Snippet: Proteins were separated by 8%–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Immunobilon-P, 0.45 mm; Millipore, Billerica, MA, USA) using the TE 77 Semi-Dry Transfer Unit (GE Healthcare Life Sciences, Buckinghamshire, UK).

    Techniques: Activation Assay, SDS Page, Standard Deviation

    Isolated collagen species from chicken and xenopus on 6% SDS-PAGE. A, chicken collagens; lane 1, acid soluble fraction; lane 2, neutral 1.0 M salt soluble fraction ; lane 3, acid 1.2 M salt fraction; lane 4, neutral salt insoluble fraction; lane 5, reduced neutral salt insoluble fraction. B, xenopus collagens; lane 1, acid soluble fraction; lane 2, neutral 1.0 M salt soluble fraction; lane 3, acid 1.2 M salt fraction; lane 4, neutral salt insoluble fraction; lane 5, reduced neutral salt insoluble fraction. β11(I) and β12(I) are cross-linked α1-α1 and α1-α2 chain dimers, respectively.

    Journal: PLoS ONE

    Article Title: Insights on the Evolution of Prolyl 3-Hydroxylation Sites from Comparative Analysis of Chicken and Xenopus Fibrillar Collagens

    doi: 10.1371/journal.pone.0019336

    Figure Lengend Snippet: Isolated collagen species from chicken and xenopus on 6% SDS-PAGE. A, chicken collagens; lane 1, acid soluble fraction; lane 2, neutral 1.0 M salt soluble fraction ; lane 3, acid 1.2 M salt fraction; lane 4, neutral salt insoluble fraction; lane 5, reduced neutral salt insoluble fraction. B, xenopus collagens; lane 1, acid soluble fraction; lane 2, neutral 1.0 M salt soluble fraction; lane 3, acid 1.2 M salt fraction; lane 4, neutral salt insoluble fraction; lane 5, reduced neutral salt insoluble fraction. β11(I) and β12(I) are cross-linked α1-α1 and α1-α2 chain dimers, respectively.

    Article Snippet: Protein samples were analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Blue G-250 (Sigma-Aldrich) .

    Techniques: Isolation, SDS Page

    A) Image of cellular targets of EPO in mouse brain separated by SDS-PAGE in a 12 % gel. Gel was stained by Silver. B)Image of cellular targets of EPO in mouse brain separated by IEF in a 7 cm IPG strip containing nonlinear pH gradient 3–10 followed by two-dimensional gel electrophoresis. Protein detection was by Silver-staining

    Journal: Iranian Journal of Basic Medical Sciences

    Article Title: Recognition and characterization of Erythropoietin binding-proteins in the brain of mice

    doi:

    Figure Lengend Snippet: A) Image of cellular targets of EPO in mouse brain separated by SDS-PAGE in a 12 % gel. Gel was stained by Silver. B)Image of cellular targets of EPO in mouse brain separated by IEF in a 7 cm IPG strip containing nonlinear pH gradient 3–10 followed by two-dimensional gel electrophoresis. Protein detection was by Silver-staining

    Article Snippet: Separation of target proteins on SDS-PAGE Freeze dried elutes, from IP, step were dissolved in 20 μl of 2X SDS sample buffer, samples were incubated in boiling water for 5 min and then subjected to SDS-PAGE electrophoresis (BioRad).

    Techniques: SDS Page, Staining, Electrofocusing, Stripping Membranes, Two-Dimensional Gel Electrophoresis, Electrophoresis, Silver Staining

    Sucrose gradient sedimentation profiles of the NUDA and NUDG proteins. A sample from each of the top nine fractions from the gradient was subjected to SDS-PAGE and Western blotted with antibody against the nudA CDHC or the nudG 8-kD CDLC.

    Journal: The Journal of Cell Biology

    Article Title: The "8-kD" Cytoplasmic Dynein Light Chain Is Required for Nuclear Migration and for Dynein Heavy Chain Localization in Aspergillus nidulans

    doi:

    Figure Lengend Snippet: Sucrose gradient sedimentation profiles of the NUDA and NUDG proteins. A sample from each of the top nine fractions from the gradient was subjected to SDS-PAGE and Western blotted with antibody against the nudA CDHC or the nudG 8-kD CDLC.

    Article Snippet: The immunoprecipitate was resuspended in Laemmli sample buffer, subjected to electrophoresis on a 4–20% gradient SDS-PAGE gel (Bio-Rad Laboratories, Richmond, CA) and transferred to an Immobilon-P membrane ( Millipore Corp. , Bedford, MA).

    Techniques: Sedimentation, SDS Page, Western Blot

    LEF1 protein expression is decreased in hMSC-AML. (A) Western blot analysis of LEF1. Protein extracts (30 μg) from hMSC-HD (1–8) and hMSC-AML (9–16) were separated by SDS-PAGE and probed with an LEF1 antibody. Ponceau staining was used as a loading control. (B) Representative graphic of the electrophoresis results confirming the decrease in LEF1 expression in hMSC-AML compared to that in hMSC-HD. The bars indicate the mean protein levels (± standard deviation). * P

    Journal: Translational Oncology

    Article Title: Canonical WNT Signaling Pathway is Altered in Mesenchymal Stromal Cells From Acute Myeloid Leukemia Patients And Is Implicated in BMP4 Down-Regulation

    doi: 10.1016/j.tranon.2019.01.003

    Figure Lengend Snippet: LEF1 protein expression is decreased in hMSC-AML. (A) Western blot analysis of LEF1. Protein extracts (30 μg) from hMSC-HD (1–8) and hMSC-AML (9–16) were separated by SDS-PAGE and probed with an LEF1 antibody. Ponceau staining was used as a loading control. (B) Representative graphic of the electrophoresis results confirming the decrease in LEF1 expression in hMSC-AML compared to that in hMSC-HD. The bars indicate the mean protein levels (± standard deviation). * P

    Article Snippet: Protein extracts (30 μg) were separated with 10% SDS-PAGE gel electrophoresis, transferred to nitrocellulose membranes (Bio-Rad), and incubated with anti-LEF1 antibody (Santa Cruz Biotechnology) at 4°C overnight, followed by incubation with the appropriate secondary antibody at room temperature (RT) for 2 h. Rouge Ponceau staining was used as the loading control.

    Techniques: Expressing, Western Blot, SDS Page, Staining, Electrophoresis, Standard Deviation

    p53 oligomerization is decreased in p53 334H/H mouse tissues. A , liver tissue lysates were cross-linked with glutaraldehyde, resolved in SDS-PAGE gel, and immunoblotted. Note p53 antibody specificity demonstrated by lack of immunoreactivity in the p53 −/− sample. Protein standards are in kD. Triplicate lanes of each genotype represent liver samples from 3 separate mice. B, fraction of p53 oligomers (T, tetramer; D, dimer; M, monomer) within each lane of immunoblot (A) quantified by densitometry and compared with the respective wild-type oligomer ( n = 3). C , p53 immunoblot of cross-linked liver lysates obtained from mice 6 h after p53 induction by doxorubicin treatment. Duplicate lanes of each genotype represent liver samples from 2 separate mice. D , p53 binding to the p53 response element (p53RE) of p21 in γ-irradiated (γ-IR) mouse liver. ChIP was performed using nonspecific IgG or anti-p53 antibody. p53RE binding is shown relative to wild-type non-specific IgG samples ( n = 3). E, induction of p21 mRNA in the indicated tissues by γ-IR quantified by RT-PCR ( n = 3). Levels were normalized relative to a housekeeping gene TIF . Bone marrow (BM); small intestine. p53 R334 genotypes: wild-type ( R/R ); heterozygous mutant ( R/H ); homozygous mutant ( H/H ); and p53 null ( −/− ). Values are mean ± SD. Compared to wild-type samples within each group, statistical differences were tested by 1-way ANOVA. * P

    Journal: Cancer research

    Article Title: Mouse homolog of the human TP53 R337H mutation reveals its role in tumorigenesis

    doi: 10.1158/0008-5472.CAN-18-0016

    Figure Lengend Snippet: p53 oligomerization is decreased in p53 334H/H mouse tissues. A , liver tissue lysates were cross-linked with glutaraldehyde, resolved in SDS-PAGE gel, and immunoblotted. Note p53 antibody specificity demonstrated by lack of immunoreactivity in the p53 −/− sample. Protein standards are in kD. Triplicate lanes of each genotype represent liver samples from 3 separate mice. B, fraction of p53 oligomers (T, tetramer; D, dimer; M, monomer) within each lane of immunoblot (A) quantified by densitometry and compared with the respective wild-type oligomer ( n = 3). C , p53 immunoblot of cross-linked liver lysates obtained from mice 6 h after p53 induction by doxorubicin treatment. Duplicate lanes of each genotype represent liver samples from 2 separate mice. D , p53 binding to the p53 response element (p53RE) of p21 in γ-irradiated (γ-IR) mouse liver. ChIP was performed using nonspecific IgG or anti-p53 antibody. p53RE binding is shown relative to wild-type non-specific IgG samples ( n = 3). E, induction of p21 mRNA in the indicated tissues by γ-IR quantified by RT-PCR ( n = 3). Levels were normalized relative to a housekeeping gene TIF . Bone marrow (BM); small intestine. p53 R334 genotypes: wild-type ( R/R ); heterozygous mutant ( R/H ); homozygous mutant ( H/H ); and p53 null ( −/− ). Values are mean ± SD. Compared to wild-type samples within each group, statistical differences were tested by 1-way ANOVA. * P

    Article Snippet: The protein samples were resolved on 4–20% gradient SDS/PAGE gels (Bio-Rad Laboratories) and immunoblotted with anti-p53 mouse monoclonal antibody (#2524, Cell Signaling Technology).

    Techniques: SDS Page, Mouse Assay, Binding Assay, Irradiation, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Mutagenesis

    Western blot demonstrating antigenicity of the S. mansoni proteins expressed in the WGL system. Non-purified wheat germ extracts containing in vitro translated IPSE/alpha-1, SmTAL1 or SmTAL2 were separated in a 4–20% SDS-PAGE gel and blotted onto NCM. Separate strips of NCM were treated with anti-TAL1 rabbit serum, anti-TAL2 rabbit serum or anti-IPSE/alpha-1 mouse monoclonal antibody. The negative control (neg. control) was incubated without primary antibody/serum, but with secondary antibody. Membranes were imaged using chemiluminescence and a Fujifilm LAS-4000.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Use of Humanised Rat Basophilic Leukaemia Cell Line RS-ATL8 for the Assessment of Allergenicity of Schistosoma mansoni Proteins

    doi: 10.1371/journal.pntd.0003124

    Figure Lengend Snippet: Western blot demonstrating antigenicity of the S. mansoni proteins expressed in the WGL system. Non-purified wheat germ extracts containing in vitro translated IPSE/alpha-1, SmTAL1 or SmTAL2 were separated in a 4–20% SDS-PAGE gel and blotted onto NCM. Separate strips of NCM were treated with anti-TAL1 rabbit serum, anti-TAL2 rabbit serum or anti-IPSE/alpha-1 mouse monoclonal antibody. The negative control (neg. control) was incubated without primary antibody/serum, but with secondary antibody. Membranes were imaged using chemiluminescence and a Fujifilm LAS-4000.

    Article Snippet: Samples were heated for 3 minutes at 70°C, run on 4–20% SDS-PAGE gradient gels (BioRad, UK), under reducing conditions and imaged with a laser-based fluorescent gel scanner (Fujifilm LAS-4000 319 Imaging System).

    Techniques: Western Blot, Purification, In Vitro, SDS Page, Negative Control, Incubation

    In gel detection of five different S. mansoni antigens expressed in vitro using WGL. SPO-1: Smp_113760; GST-26k: Smp_102070; TSP-1, extracellular loop 2: Smp_095630; IPSE/alpha-1: Smp_112110; SmTAL1: Smp_045200; SmTAL2: Smp_086480. Details of sequences and expected molecular weights are given in the Supplementary Data in Table S3 . Success of translation was monitored by incorporation of BODIPY-labelled fluorescent Lysine in separate aliquots during translation. Samples were run on 4–20% SDS-PAGE gradient gels under reducing conditions and imaged in a Fujifilm LAS-4000. The left lanes show the wheat germ lysate control without template DNA, either without (−K) or with Lysine incorporation (+K), indicating fluorescent components produced during in vitro translation from endogenous mRNA.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Use of Humanised Rat Basophilic Leukaemia Cell Line RS-ATL8 for the Assessment of Allergenicity of Schistosoma mansoni Proteins

    doi: 10.1371/journal.pntd.0003124

    Figure Lengend Snippet: In gel detection of five different S. mansoni antigens expressed in vitro using WGL. SPO-1: Smp_113760; GST-26k: Smp_102070; TSP-1, extracellular loop 2: Smp_095630; IPSE/alpha-1: Smp_112110; SmTAL1: Smp_045200; SmTAL2: Smp_086480. Details of sequences and expected molecular weights are given in the Supplementary Data in Table S3 . Success of translation was monitored by incorporation of BODIPY-labelled fluorescent Lysine in separate aliquots during translation. Samples were run on 4–20% SDS-PAGE gradient gels under reducing conditions and imaged in a Fujifilm LAS-4000. The left lanes show the wheat germ lysate control without template DNA, either without (−K) or with Lysine incorporation (+K), indicating fluorescent components produced during in vitro translation from endogenous mRNA.

    Article Snippet: Samples were heated for 3 minutes at 70°C, run on 4–20% SDS-PAGE gradient gels (BioRad, UK), under reducing conditions and imaged with a laser-based fluorescent gel scanner (Fujifilm LAS-4000 319 Imaging System).

    Techniques: In Vitro, SDS Page, Produced

    Co-immunoprecipitation of the selective recruitment of Csk and pSHP-2 to various 1.1b WT CYT constructs following co-crosslinking with 2.6b ITAM CYT . AD293 cells (3 × 10 5 ) co-expressing 2.6b ITAM CYT and the various 1.1b CYT constructs were incubated at 37 °C for 8 min with 3 µm magnetic beads (3 × 10 6 ) opsonized with the indicated mAbs and/or isotype IgG1 (’+’ and ‘−’ indicate the presence and absence of corresponding antibodies on beads, respectively). Cells were immediately lysed on ice for 30 min and then magnetic beads were separated, washed three times, and eluted before separating bead-bound proteins using SDS-PAGE. Separated proteins were transferred to nitrocellulose membranes and then probed with α-FLAG and α-HA mAbs ( A ) to verify successful pull-down of the various 1.1b CYT constructs (top membrane) and 2.6b ITAM CYT (bottom membrane). Co-immunoprecipitation of potential effector molecules ( B ) was further examined by probing membranes with α-Csk, α-pSHP-2, α-PTEN, and α-SHIP2 mAbs. For the bottom two panels in ( B ), a whole cell lysate (WCL) sample is included to show that these molecules are present in AD293 lysates. Blots shown are representative examples of three independent experiments.

    Journal: International Journal of Molecular Sciences

    Article Title: A Fish Leukocyte Immune-Type Receptor Uses a Novel Intracytoplasmic Tail Networking Mechanism to Cross-Inhibit the Phagocytic Response

    doi: 10.3390/ijms21145146

    Figure Lengend Snippet: Co-immunoprecipitation of the selective recruitment of Csk and pSHP-2 to various 1.1b WT CYT constructs following co-crosslinking with 2.6b ITAM CYT . AD293 cells (3 × 10 5 ) co-expressing 2.6b ITAM CYT and the various 1.1b CYT constructs were incubated at 37 °C for 8 min with 3 µm magnetic beads (3 × 10 6 ) opsonized with the indicated mAbs and/or isotype IgG1 (’+’ and ‘−’ indicate the presence and absence of corresponding antibodies on beads, respectively). Cells were immediately lysed on ice for 30 min and then magnetic beads were separated, washed three times, and eluted before separating bead-bound proteins using SDS-PAGE. Separated proteins were transferred to nitrocellulose membranes and then probed with α-FLAG and α-HA mAbs ( A ) to verify successful pull-down of the various 1.1b CYT constructs (top membrane) and 2.6b ITAM CYT (bottom membrane). Co-immunoprecipitation of potential effector molecules ( B ) was further examined by probing membranes with α-Csk, α-pSHP-2, α-PTEN, and α-SHIP2 mAbs. For the bottom two panels in ( B ), a whole cell lysate (WCL) sample is included to show that these molecules are present in AD293 lysates. Blots shown are representative examples of three independent experiments.

    Article Snippet: Proteins were separated via 10% SDS-PAGE gel and transferred to nitrocellulose membranes (Bio-Rad) that were probed with an HRP-conjugated α-HA mAb (1:1000) and an HRP-conjugated α-FLAG mAb (1:1000; Thermo Fisher Scientific) at 4 °C overnight to verify pull-down of the various epitope-tagged IpLITR constructs.

    Techniques: Immunoprecipitation, Construct, Expressing, Incubation, Magnetic Beads, SDS Page

    New monoclonal antibodies visualize parkin in ageing human midbrain. (a-c) Characterization of four murine, monoclonal antibodies (of IgG 2 isotype; clone-B, -E, -D, and -G) in three different assays: (a) against recombinant (r-), full-length, untagged, wildtype (WT) human parkin using non-denaturing slot blots (100ng/slot) of original antigen as well as truncated r-parkin 321-465 and full-length, untagged, human r-DJ-1; (b) against human brain lysates (SDS fractions from control and PRKN- linked ARPD cases) using non-denaturing dot blots; and (c) by denaturing SDS/PAGE and Western blotting of extracts from cortical specimens of a control brain and a parkin-deficient ARPD case. Screening by these three methods as well as by cell-based microscopy (using indirect immunofluorescence) revealed specific staining for four anti-parkin clones (-B, -E, -D and -G), which was conformation-dependent for clone-E. List of epitopes within the sequence of human parkin, as recognized by clones -B, -E, -D, and -G and informed by overlapping screening with 7-12 amino acid-long peptides covering full-length, human parkin. Note that the clone E epitope is conformational, comprised of the three regions indicated.

    Journal: bioRxiv

    Article Title: Oxidative Modifications of Parkin Underlie its Selective Neuroprotection in Adult Human Brain

    doi: 10.1101/2020.02.19.953034

    Figure Lengend Snippet: New monoclonal antibodies visualize parkin in ageing human midbrain. (a-c) Characterization of four murine, monoclonal antibodies (of IgG 2 isotype; clone-B, -E, -D, and -G) in three different assays: (a) against recombinant (r-), full-length, untagged, wildtype (WT) human parkin using non-denaturing slot blots (100ng/slot) of original antigen as well as truncated r-parkin 321-465 and full-length, untagged, human r-DJ-1; (b) against human brain lysates (SDS fractions from control and PRKN- linked ARPD cases) using non-denaturing dot blots; and (c) by denaturing SDS/PAGE and Western blotting of extracts from cortical specimens of a control brain and a parkin-deficient ARPD case. Screening by these three methods as well as by cell-based microscopy (using indirect immunofluorescence) revealed specific staining for four anti-parkin clones (-B, -E, -D and -G), which was conformation-dependent for clone-E. List of epitopes within the sequence of human parkin, as recognized by clones -B, -E, -D, and -G and informed by overlapping screening with 7-12 amino acid-long peptides covering full-length, human parkin. Note that the clone E epitope is conformational, comprised of the three regions indicated.

    Article Snippet: Protein staining methods All proteins were separated on pre-cast 4-12 % Bis-Tris SDS-PAGE gels (NPO321BOX, NPO322BOX, NPO336BOX) from Invitrogen using MES running buffer (50mM MES, 50mM Tris, 1mM EDTA and 0.1 % SDS, pH 7.3) and Laemmli loading buffer (10% SDS, 20% glycerol, 0.1% bromophenol blue, 0.125M Tris HCl, 200mM DTT or β-mercaptoethanol).

    Techniques: Recombinant, SDS Page, Western Blot, Microscopy, Immunofluorescence, Staining, Clone Assay, Sequencing

    Parkin transitions from a soluble to an aggregated state in adult human midbrain. (a) Representative Western blots of parkin, DJ-1, and LC3B distribution in human cortex, S. nigra (SN) and red nucleus (RN) brain specimens that had been serially fractionated into Tris-NaCl buffer-soluble (TS), Triton X-100-soluble (TX), 2% SDS-soluble (SDS) extracts and the pellet (P) lysed in 30% SDS-containing buffer. Lysates from PRKN -linked Parkinson disease (ARPD) brain and recombinant, human parkin (r-parkin) are included. (b-c) Relative distribution of parkin signal within each fraction for (b) cortex and (c) midbrain grouped by age ranges: young (Y; ≤ 20y; n=13); mid (M, > 20y,

    Journal: bioRxiv

    Article Title: Oxidative Modifications of Parkin Underlie its Selective Neuroprotection in Adult Human Brain

    doi: 10.1101/2020.02.19.953034

    Figure Lengend Snippet: Parkin transitions from a soluble to an aggregated state in adult human midbrain. (a) Representative Western blots of parkin, DJ-1, and LC3B distribution in human cortex, S. nigra (SN) and red nucleus (RN) brain specimens that had been serially fractionated into Tris-NaCl buffer-soluble (TS), Triton X-100-soluble (TX), 2% SDS-soluble (SDS) extracts and the pellet (P) lysed in 30% SDS-containing buffer. Lysates from PRKN -linked Parkinson disease (ARPD) brain and recombinant, human parkin (r-parkin) are included. (b-c) Relative distribution of parkin signal within each fraction for (b) cortex and (c) midbrain grouped by age ranges: young (Y; ≤ 20y; n=13); mid (M, > 20y,

    Article Snippet: Protein staining methods All proteins were separated on pre-cast 4-12 % Bis-Tris SDS-PAGE gels (NPO321BOX, NPO322BOX, NPO336BOX) from Invitrogen using MES running buffer (50mM MES, 50mM Tris, 1mM EDTA and 0.1 % SDS, pH 7.3) and Laemmli loading buffer (10% SDS, 20% glycerol, 0.1% bromophenol blue, 0.125M Tris HCl, 200mM DTT or β-mercaptoethanol).

    Techniques: Western Blot, Recombinant

    Parkin’s secondary structure is altered by redox stress. (a) Silver staining of r-parkin in soluble (supernatant) and insoluble (pellet) phases following exposure to increasing concentrations of H 2 O 2 (0-2mM) and run under non-reducing conditions. Monomer (*) and high M r weight (HMW) r-parkin species are indicated. (b) Silver stained gel of r-parkin exposed to H 2 O 2 (10 mM), followed by treatment with increasing concentrations of DTT (0-100 mM) prior to centrifugation and loading of the supernatant onto SDS-PAGE. (c,d) Circular dichroism spectra of soluble, untreated r-parkin at (c) T=0 and (d) soluble (black line) and aggregated (red line) states following incubation at 37°C for T=5 days. The protein secondary structure shifts from a predominant appearance of α-helix dominated state, as demonstrated by the positive band at 193 nm and negative bands 208 nm (black lines), to the appearance of β-pleated sheet formation, as demonstrated by negative bands at 218 nm and a rise in molar ellipticity with positive bands at 195 nm (red lines) during spontaneous oxidation. (e-f) Quantitative analyses of IAA-modified cysteines captured by LC-MS/MS for (e) untreated vs. H 2 O 2 -exposed r-parkin, and (f) soluble compared to insoluble (pellet) fractions. Each data point represents the log2-transformed total IAA-signal intensities of single cysteine residues (n=3 runs for each). The cysteine pool is shown with the mean ± SEM; significance **p

    Journal: bioRxiv

    Article Title: Oxidative Modifications of Parkin Underlie its Selective Neuroprotection in Adult Human Brain

    doi: 10.1101/2020.02.19.953034

    Figure Lengend Snippet: Parkin’s secondary structure is altered by redox stress. (a) Silver staining of r-parkin in soluble (supernatant) and insoluble (pellet) phases following exposure to increasing concentrations of H 2 O 2 (0-2mM) and run under non-reducing conditions. Monomer (*) and high M r weight (HMW) r-parkin species are indicated. (b) Silver stained gel of r-parkin exposed to H 2 O 2 (10 mM), followed by treatment with increasing concentrations of DTT (0-100 mM) prior to centrifugation and loading of the supernatant onto SDS-PAGE. (c,d) Circular dichroism spectra of soluble, untreated r-parkin at (c) T=0 and (d) soluble (black line) and aggregated (red line) states following incubation at 37°C for T=5 days. The protein secondary structure shifts from a predominant appearance of α-helix dominated state, as demonstrated by the positive band at 193 nm and negative bands 208 nm (black lines), to the appearance of β-pleated sheet formation, as demonstrated by negative bands at 218 nm and a rise in molar ellipticity with positive bands at 195 nm (red lines) during spontaneous oxidation. (e-f) Quantitative analyses of IAA-modified cysteines captured by LC-MS/MS for (e) untreated vs. H 2 O 2 -exposed r-parkin, and (f) soluble compared to insoluble (pellet) fractions. Each data point represents the log2-transformed total IAA-signal intensities of single cysteine residues (n=3 runs for each). The cysteine pool is shown with the mean ± SEM; significance **p

    Article Snippet: Protein staining methods All proteins were separated on pre-cast 4-12 % Bis-Tris SDS-PAGE gels (NPO321BOX, NPO322BOX, NPO336BOX) from Invitrogen using MES running buffer (50mM MES, 50mM Tris, 1mM EDTA and 0.1 % SDS, pH 7.3) and Laemmli loading buffer (10% SDS, 20% glycerol, 0.1% bromophenol blue, 0.125M Tris HCl, 200mM DTT or β-mercaptoethanol).

    Techniques: Silver Staining, Staining, Centrifugation, SDS Page, Incubation, Modification, Liquid Chromatography with Mass Spectroscopy, Transformation Assay

    Redox potentials of the cysteine pairs in the P1 and P2 loops of α-DsbB. ( A ) Redox equilibria of α-DsbB [CCSS] and α-DsbB [SSCC]. After equilibration in redox buffers of varying [DTTred]/[DTTox] ratios for 24 h, samples were treated with 10% TCA and free cysteines were labeled with AMS. Reduced and oxidized proteins were separated by 12% SDS-PAGE under non-reducing conditions. ( B ) Redox potentials of α-DsbB [CCSS] and α-DsbB [SSCC]. The fractions of α-DsbB [CCSS] or α-DsbB [SSCC] in the reduced state were calculated using ImageJ [36] and equilibrium constants were plotted against the fraction of the reduced state in PRISM® using the one phase decay option. The data presented are the standard mean and error from duplicate measurements.

    Journal: PLoS ONE

    Article Title: The ?-Proteobacteria Wolbachia pipientis Protein Disulfide Machinery Has a Regulatory Mechanism Absent in γ-Proteobacteria

    doi: 10.1371/journal.pone.0081440

    Figure Lengend Snippet: Redox potentials of the cysteine pairs in the P1 and P2 loops of α-DsbB. ( A ) Redox equilibria of α-DsbB [CCSS] and α-DsbB [SSCC]. After equilibration in redox buffers of varying [DTTred]/[DTTox] ratios for 24 h, samples were treated with 10% TCA and free cysteines were labeled with AMS. Reduced and oxidized proteins were separated by 12% SDS-PAGE under non-reducing conditions. ( B ) Redox potentials of α-DsbB [CCSS] and α-DsbB [SSCC]. The fractions of α-DsbB [CCSS] or α-DsbB [SSCC] in the reduced state were calculated using ImageJ [36] and equilibrium constants were plotted against the fraction of the reduced state in PRISM® using the one phase decay option. The data presented are the standard mean and error from duplicate measurements.

    Article Snippet: To identify protein-containing fractions, 15 µL samples from peak fractions were applied to a 4–12% Bis-Tris SDS PAGE (Life Technologies, USA).

    Techniques: Labeling, Affinity Magnetic Separation, SDS Page

    Spy Go from mammalian expression. a HEK293T cells were transfected with the extracellular region of EpCAM fused to SpyTag and a His-tag (EpCAM-SpyTag). EpCAM-SpyTag was purified from the clarified cell supernatant using Spy Go. Fractions were analyzed by SDS-PAGE with Coomassie staining. T: total pooled elutions. Resin: resin post-elution. b Ni-NTA purification of EpCAM-SpyTag as in a . c Spy Go purification of CyRPA-SpyTag from Expi293HEK cells as in a

    Journal: Nature Communications

    Article Title: Spy Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox

    doi: 10.1038/s41467-019-09678-w

    Figure Lengend Snippet: Spy Go from mammalian expression. a HEK293T cells were transfected with the extracellular region of EpCAM fused to SpyTag and a His-tag (EpCAM-SpyTag). EpCAM-SpyTag was purified from the clarified cell supernatant using Spy Go. Fractions were analyzed by SDS-PAGE with Coomassie staining. T: total pooled elutions. Resin: resin post-elution. b Ni-NTA purification of EpCAM-SpyTag as in a . c Spy Go purification of CyRPA-SpyTag from Expi293HEK cells as in a

    Article Snippet: Reaction was quenched with SDS-PAGE loading buffer [0.23 M Tris HCl pH 6.8, 24% (v/v) glycerol, 120 μM bromophenol blue, 0.23 M SDS, 100 mM 2-mercaptoethanol] and heating at 95 °C for 5 min in a Bio-Rad C1000 thermal cycler.

    Techniques: Expressing, Transfection, Purification, SDS Page, Staining

    Oligomer panel tested for cancer cell killing. a Cartoon depicting depletion of free SpyTag-ligand using Spy Go resin. b Coupling of SpyTag-fusion to coiled coil series. αDR5-SpyTag was incubated with each SpyCatcher002-coiled coil and analyzed by SDS-PAGE with Coomassie staining. In Recaptured lanes, excess αDR5-SpyTag was removed from the coiled coil conjugate by an additional passage through Spy Go resin. c Dose-response curve of MDA-MB-231 cell viability when treated with different concentrations of αDR5 conjugated to each SpyCatcher002-oligomer platforms. The line at 50% cell viability shows the cut-off for EC 50 calculation. The x -axis is normalized to the concentration of αDR5 monomer. Error bars represent mean ±1 s.d., n = 2. d MDA-MB-231 viability upon incubation as in c with the building blocks of αDR5 alone or coiled coils alone. Error bars represent mean ± 1 s.d., n = 3

    Journal: Nature Communications

    Article Title: Spy Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox

    doi: 10.1038/s41467-019-09678-w

    Figure Lengend Snippet: Oligomer panel tested for cancer cell killing. a Cartoon depicting depletion of free SpyTag-ligand using Spy Go resin. b Coupling of SpyTag-fusion to coiled coil series. αDR5-SpyTag was incubated with each SpyCatcher002-coiled coil and analyzed by SDS-PAGE with Coomassie staining. In Recaptured lanes, excess αDR5-SpyTag was removed from the coiled coil conjugate by an additional passage through Spy Go resin. c Dose-response curve of MDA-MB-231 cell viability when treated with different concentrations of αDR5 conjugated to each SpyCatcher002-oligomer platforms. The line at 50% cell viability shows the cut-off for EC 50 calculation. The x -axis is normalized to the concentration of αDR5 monomer. Error bars represent mean ±1 s.d., n = 2. d MDA-MB-231 viability upon incubation as in c with the building blocks of αDR5 alone or coiled coils alone. Error bars represent mean ± 1 s.d., n = 3

    Article Snippet: Reaction was quenched with SDS-PAGE loading buffer [0.23 M Tris HCl pH 6.8, 24% (v/v) glycerol, 120 μM bromophenol blue, 0.23 M SDS, 100 mM 2-mercaptoethanol] and heating at 95 °C for 5 min in a Bio-Rad C1000 thermal cycler.

    Techniques: Incubation, SDS Page, Staining, Multiple Displacement Amplification, Concentration Assay

    Design of SpyDock. a All small residues except D blocked SpyCatcher reaction. E77 was mutated to the indicated residue and incubated with SpyTag-MBP for 24 h, before boiling in SDS and analysis by SDS-PAGE with Coomassie staining. b Additional mutations in SpyCatcher enhanced Spy Go purification. The original SpyCatcher, the evolved SpyCatcher002 or SpyCatcher2.1 bearing the E77A mutation were compared for capture and elution of SpyTag-MBP (SDS-PAGE with Coomassie staining). Further E77X mutations were similarly explored on SpyCatcher2.1. c Positions of mutations screened for SpyDock acceleration, based on Protein Data Bank 4MLI; CnaB2 triad represented in spheres, anchoring sites in yellow, and SpyCatcher2.1 accelerating mutations in green. d S49C resin anchoring allowed efficient SpyDock purification. SpyCatcher2.1 E77A S49C was coupled to SulfoLink resin and tested for SpyTag-MBP capture and release (SDS-PAGE with Coomassie staining)

    Journal: Nature Communications

    Article Title: Spy Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox

    doi: 10.1038/s41467-019-09678-w

    Figure Lengend Snippet: Design of SpyDock. a All small residues except D blocked SpyCatcher reaction. E77 was mutated to the indicated residue and incubated with SpyTag-MBP for 24 h, before boiling in SDS and analysis by SDS-PAGE with Coomassie staining. b Additional mutations in SpyCatcher enhanced Spy Go purification. The original SpyCatcher, the evolved SpyCatcher002 or SpyCatcher2.1 bearing the E77A mutation were compared for capture and elution of SpyTag-MBP (SDS-PAGE with Coomassie staining). Further E77X mutations were similarly explored on SpyCatcher2.1. c Positions of mutations screened for SpyDock acceleration, based on Protein Data Bank 4MLI; CnaB2 triad represented in spheres, anchoring sites in yellow, and SpyCatcher2.1 accelerating mutations in green. d S49C resin anchoring allowed efficient SpyDock purification. SpyCatcher2.1 E77A S49C was coupled to SulfoLink resin and tested for SpyTag-MBP capture and release (SDS-PAGE with Coomassie staining)

    Article Snippet: Reaction was quenched with SDS-PAGE loading buffer [0.23 M Tris HCl pH 6.8, 24% (v/v) glycerol, 120 μM bromophenol blue, 0.23 M SDS, 100 mM 2-mercaptoethanol] and heating at 95 °C for 5 min in a Bio-Rad C1000 thermal cycler.

    Techniques: Incubation, SDS Page, Staining, Purification, Mutagenesis

    Spy Go from bacterial expression. a SpyTag-MBP was purified from E. coli clarified lysate by Spy Go. Protein: input SpyTag-MBP protein. T: total pooled elutions. Purity of T was determined by densitometry (right); gray represents background lane intensity (mean ± 1 s.d., n = 3). b Ni-NTA purification of SpyTag-MBP from the same lysate via its His-tag. c Spy Go purification of scPvuII-SpyTag (SpyTag at an internal loop, shown schematically) from bacterial lysate. d Spy Go purification of the nanobody αDR5-SpyTag (C-terminal fusion) from bacterial lysate. All fractions were analyzed by SDS-PAGE with Coomassie staining

    Journal: Nature Communications

    Article Title: Spy Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox

    doi: 10.1038/s41467-019-09678-w

    Figure Lengend Snippet: Spy Go from bacterial expression. a SpyTag-MBP was purified from E. coli clarified lysate by Spy Go. Protein: input SpyTag-MBP protein. T: total pooled elutions. Purity of T was determined by densitometry (right); gray represents background lane intensity (mean ± 1 s.d., n = 3). b Ni-NTA purification of SpyTag-MBP from the same lysate via its His-tag. c Spy Go purification of scPvuII-SpyTag (SpyTag at an internal loop, shown schematically) from bacterial lysate. d Spy Go purification of the nanobody αDR5-SpyTag (C-terminal fusion) from bacterial lysate. All fractions were analyzed by SDS-PAGE with Coomassie staining

    Article Snippet: Reaction was quenched with SDS-PAGE loading buffer [0.23 M Tris HCl pH 6.8, 24% (v/v) glycerol, 120 μM bromophenol blue, 0.23 M SDS, 100 mM 2-mercaptoethanol] and heating at 95 °C for 5 min in a Bio-Rad C1000 thermal cycler.

    Techniques: Expressing, Purification, SDS Page, Staining

    Immunodetection of Cr LPAAT 1 expression in C. reinhardtii cells expressing HA ‐tagged Cr LPAAT 1 using anti‐ HA antibodies. (a) Immunoblot analysis of C. reinhardtii cells expressing HA ‐tagged Cr LPAAT 1 using anti‐ HA antibodies. Two bands were detected; the upper band corresponds to the full protein (36.5 kDa), and the lower band corresponds to the mature protein (31.5 kDa) lacking the transit peptide. (b) The SDS ‐ PAGE gel was stained with blue dye (ProSieve EX Safe Stain— LONZA ) as a loading control for the immunoblot shown in (a). Twenty micrograms of total proteins were loaded onto an SDS ‐ PAGE gel, and expression of Cr LPAAT 1 was detected using anti‐ HA antibodies. A duplicate of the gel was also visualized after staining with blue dye for 1 h. OE : pLM 21‐Cr LPAAT 1‐ HA overexpressors; three transformants ( OE 1, OE 2 and OE 3) are shown.

    Journal: Plant Biotechnology Journal

    Article Title: Identification of a Chlamydomonas plastidial 2‐lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content

    doi: 10.1111/pbi.12572

    Figure Lengend Snippet: Immunodetection of Cr LPAAT 1 expression in C. reinhardtii cells expressing HA ‐tagged Cr LPAAT 1 using anti‐ HA antibodies. (a) Immunoblot analysis of C. reinhardtii cells expressing HA ‐tagged Cr LPAAT 1 using anti‐ HA antibodies. Two bands were detected; the upper band corresponds to the full protein (36.5 kDa), and the lower band corresponds to the mature protein (31.5 kDa) lacking the transit peptide. (b) The SDS ‐ PAGE gel was stained with blue dye (ProSieve EX Safe Stain— LONZA ) as a loading control for the immunoblot shown in (a). Twenty micrograms of total proteins were loaded onto an SDS ‐ PAGE gel, and expression of Cr LPAAT 1 was detected using anti‐ HA antibodies. A duplicate of the gel was also visualized after staining with blue dye for 1 h. OE : pLM 21‐Cr LPAAT 1‐ HA overexpressors; three transformants ( OE 1, OE 2 and OE 3) are shown.

    Article Snippet: Briefly, around 20 μg of protein was loaded onto a 12% Bis Tris SDS‐PAGE gel (Thermo Fisher Scientific, Waltham, MA).

    Techniques: Immunodetection, Expressing, SDS Page, Staining

    KLEIP is a 64-kDa protein and expressed ubiquitously in human adult tissues. (A) Immunoblotting analysis using an affinity-purified anti-KLEIP-N antibody. FLAG-vector (lane 1), FLAG-tagged KLEIP-N (lane 2) or -FL (lane 3) was transfected in COS cells. Cell lysates (30 μg of protein) were separated on 8–16% gradient SDS-PAGE gels and analyzed by the immunoglobulin fraction of preimmune sera (prebleed), purified anti-KLEIP-N antibody or anti-FLAG antibody. Predicted molecular sizes of KLEIP-N and -FL are 64- and 98-kDa, and the locations of these proteins are shown by arrows. Locations of molecular size markers are shown on the left of the panel. (B) Identification of endogenous KLEIP in cultured cells. Lysates of HeLa cells were analyzed by immunoblotting. Prebleed, N and C show immunoblots with the preimmune sera (for anti-KLEIP-N), anti-KLEIP-N or -C, respectively. The location of the predicted KLEIP is shown by an arrow. (C) Immunoprecipitation of KLEIP. HeLa cell lysates (390 μg of protein) were immunoprecipitated by 0.25 μg of the IgG fraction of preimmune sera (lane 1) or anti-KLEIP-N antibody (lane 2), followed by immunoblotting with IgG of preimmune sera or affinity-purified anti-KLEIP-N antibody. An arrow denotes the 64-kDa protein. Asterisks show bands of heavy and light chains of IgG. (D) Identification of endogenous KLEIP in cultured cells. Lysates (10 μg of protein in each) were separated by SDS-PAGE gels and analyzed by immunoblotting with anti-KLEIP-N. Tubulin was detected as a loading control (bottom). (E) mRNA expression of KLEIP in human tissues. PCR was performed to detect expression of KLEIP (top) or glyceraldehyde-3-phosphate dehydrogenase G3PDH (bottom). Lane 1, heart; 2, brain; 3, liver; 4, placenta; 5, lung; 6, kidney; 7, skeletal muscle; 8, pancreas; 9, spleen; 10, thymus; 11, small intestine; 12, testis; 13, ovary; and 14, colon.

    Journal: Molecular Biology of the Cell

    Article Title: Novel Kelch-like Protein, KLEIP, Is Involved in Actin Assembly at Cell-Cell Contact Sites of Madin-Darby Canine Kidney Cells

    doi: 10.1091/mbc.E03-07-0531

    Figure Lengend Snippet: KLEIP is a 64-kDa protein and expressed ubiquitously in human adult tissues. (A) Immunoblotting analysis using an affinity-purified anti-KLEIP-N antibody. FLAG-vector (lane 1), FLAG-tagged KLEIP-N (lane 2) or -FL (lane 3) was transfected in COS cells. Cell lysates (30 μg of protein) were separated on 8–16% gradient SDS-PAGE gels and analyzed by the immunoglobulin fraction of preimmune sera (prebleed), purified anti-KLEIP-N antibody or anti-FLAG antibody. Predicted molecular sizes of KLEIP-N and -FL are 64- and 98-kDa, and the locations of these proteins are shown by arrows. Locations of molecular size markers are shown on the left of the panel. (B) Identification of endogenous KLEIP in cultured cells. Lysates of HeLa cells were analyzed by immunoblotting. Prebleed, N and C show immunoblots with the preimmune sera (for anti-KLEIP-N), anti-KLEIP-N or -C, respectively. The location of the predicted KLEIP is shown by an arrow. (C) Immunoprecipitation of KLEIP. HeLa cell lysates (390 μg of protein) were immunoprecipitated by 0.25 μg of the IgG fraction of preimmune sera (lane 1) or anti-KLEIP-N antibody (lane 2), followed by immunoblotting with IgG of preimmune sera or affinity-purified anti-KLEIP-N antibody. An arrow denotes the 64-kDa protein. Asterisks show bands of heavy and light chains of IgG. (D) Identification of endogenous KLEIP in cultured cells. Lysates (10 μg of protein in each) were separated by SDS-PAGE gels and analyzed by immunoblotting with anti-KLEIP-N. Tubulin was detected as a loading control (bottom). (E) mRNA expression of KLEIP in human tissues. PCR was performed to detect expression of KLEIP (top) or glyceraldehyde-3-phosphate dehydrogenase G3PDH (bottom). Lane 1, heart; 2, brain; 3, liver; 4, placenta; 5, lung; 6, kidney; 7, skeletal muscle; 8, pancreas; 9, spleen; 10, thymus; 11, small intestine; 12, testis; 13, ovary; and 14, colon.

    Article Snippet: Samples were separated and analyzed on 8–16% gradient SDS-PAGE gels (Invitrogen) and then transferred to polyvinylidene difluoride membranes (Invitrogen).

    Techniques: Affinity Purification, Plasmid Preparation, Transfection, SDS Page, Purification, Cell Culture, Western Blot, Immunoprecipitation, Expressing, Polymerase Chain Reaction

    KLEIP forms dimers and interacts with F-actin in vitro. (A) Homodimer-formation through the BTB/POZ domain of KLEIP. MBP and MBP-KLEIP-N were cross-linked by DTSP in PBS. The cross-linkage was cleavable with DTT. Both reduced and non-reduced samples were separated on 8–16% gradient SDS-PAGE gels and then visualized by Coomassie staining. Locations of molecular makers are shown at the left. Note that MBP-dimers were not detected. Arrows indicate the predicted locations of monomeric (Mono) and dimeric KLEIP (Dimer). (B) Actin cosedimentation assays. The supernatants (S) and precipitates (P) after centrifugation at 150,000 × g were separated on 8–16% gradient SDS-PAGE gels and then visualized by Coomassie staining. MBP-KLEIP-FL (∼115 kDa) was analyzed after incubation without (lane 1) or with F-actin (lane 2). α-Actinin was used as a positive control (lane 4). Arrowheads indicate the locations of MBP-KLEIP-N and -FL. The position of actin is shown by an arrow. Locations of molecular size markers are indicated on the left.

    Journal: Molecular Biology of the Cell

    Article Title: Novel Kelch-like Protein, KLEIP, Is Involved in Actin Assembly at Cell-Cell Contact Sites of Madin-Darby Canine Kidney Cells

    doi: 10.1091/mbc.E03-07-0531

    Figure Lengend Snippet: KLEIP forms dimers and interacts with F-actin in vitro. (A) Homodimer-formation through the BTB/POZ domain of KLEIP. MBP and MBP-KLEIP-N were cross-linked by DTSP in PBS. The cross-linkage was cleavable with DTT. Both reduced and non-reduced samples were separated on 8–16% gradient SDS-PAGE gels and then visualized by Coomassie staining. Locations of molecular makers are shown at the left. Note that MBP-dimers were not detected. Arrows indicate the predicted locations of monomeric (Mono) and dimeric KLEIP (Dimer). (B) Actin cosedimentation assays. The supernatants (S) and precipitates (P) after centrifugation at 150,000 × g were separated on 8–16% gradient SDS-PAGE gels and then visualized by Coomassie staining. MBP-KLEIP-FL (∼115 kDa) was analyzed after incubation without (lane 1) or with F-actin (lane 2). α-Actinin was used as a positive control (lane 4). Arrowheads indicate the locations of MBP-KLEIP-N and -FL. The position of actin is shown by an arrow. Locations of molecular size markers are indicated on the left.

    Article Snippet: Samples were separated and analyzed on 8–16% gradient SDS-PAGE gels (Invitrogen) and then transferred to polyvinylidene difluoride membranes (Invitrogen).

    Techniques: In Vitro, SDS Page, Staining, Centrifugation, Incubation, Positive Control

    SDS-PAGE silver staining of mTHPC-loaded EGa1-conjugated micelles (EGa1-P n micelles) obtained after 10 washes with PBS following the overnight conjugation of micelles with deprotected EGa1-SATA. Native EGa1 was used as a control. The red arrow indicates a band of EGa1 with one polymer chain conjugated.

    Journal: Molecular Pharmaceutics

    Article Title: EGFR-Targeted Nanobody Functionalized Polymeric Micelles Loaded with mTHPC for Selective Photodynamic Therapy

    doi: 10.1021/acs.molpharmaceut.9b01280

    Figure Lengend Snippet: SDS-PAGE silver staining of mTHPC-loaded EGa1-conjugated micelles (EGa1-P n micelles) obtained after 10 washes with PBS following the overnight conjugation of micelles with deprotected EGa1-SATA. Native EGa1 was used as a control. The red arrow indicates a band of EGa1 with one polymer chain conjugated.

    Article Snippet: Briefly, samples were incubated with lithium dodecyl sulfate (LDS) running buffer (Bolt, Novex, Life Technologies) under reducing conditions at 80 °C for 10 min and then loaded into SDS-PAGE gel (Bolt, 4–12% Bis-Tris Plus 1.0 mm × 10 wells, Invitrogen Thermo Fisher Scientific).

    Techniques: SDS Page, Silver Staining, Conjugation Assay