Structured Review

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The effect of salt treatment on PTOX localization in thylakoid membrane of Arabidopsis and Eutrema plants subjected to 0 and 100 and 0 and 250 mM NaCl, respectively. Chloroplasts isolated 10 d after initiating salt treatment were fractionated into thylakoid membranes (T), granal thylakoid (G), stromal lamellae (L), and stroma (S). Protein samples (10 µ g) were separated by SDS/PAGE, followed by transfer to <t>PVDF</t> membrane, and immunoblotted with antibodies specific for PTOX ( A ). Purity of the fractions was controlled in Arabidopsis and Eutrema by separation and immunoblotting of the samples (5 μg) with antibodies specific for representative polypeptides ( B ). Coomassie brillant blue-stained SDS/PAGE gels of the thylakoid membrane fractions with chlorophyll a / b ratios given below each fraction ( C ). Linearity of the anti-PTOX immunodetection was ensured with respect to the amount of protein per lane. <t>Immunoblot</t> of thylakoid membranes isolated from the control plants of wild-type Eutrema presented ( D ).
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Images

1) Product Images from "Plastid terminal oxidase requires translocation to the grana stacks to act as a sink for electron transport"

Article Title: Plastid terminal oxidase requires translocation to the grana stacks to act as a sink for electron transport

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

doi: 10.1073/pnas.1719070115

The effect of salt treatment on PTOX localization in thylakoid membrane of Arabidopsis and Eutrema plants subjected to 0 and 100 and 0 and 250 mM NaCl, respectively. Chloroplasts isolated 10 d after initiating salt treatment were fractionated into thylakoid membranes (T), granal thylakoid (G), stromal lamellae (L), and stroma (S). Protein samples (10 µ g) were separated by SDS/PAGE, followed by transfer to PVDF membrane, and immunoblotted with antibodies specific for PTOX ( A ). Purity of the fractions was controlled in Arabidopsis and Eutrema by separation and immunoblotting of the samples (5 μg) with antibodies specific for representative polypeptides ( B ). Coomassie brillant blue-stained SDS/PAGE gels of the thylakoid membrane fractions with chlorophyll a / b ratios given below each fraction ( C ). Linearity of the anti-PTOX immunodetection was ensured with respect to the amount of protein per lane. Immunoblot of thylakoid membranes isolated from the control plants of wild-type Eutrema presented ( D ).
Figure Legend Snippet: The effect of salt treatment on PTOX localization in thylakoid membrane of Arabidopsis and Eutrema plants subjected to 0 and 100 and 0 and 250 mM NaCl, respectively. Chloroplasts isolated 10 d after initiating salt treatment were fractionated into thylakoid membranes (T), granal thylakoid (G), stromal lamellae (L), and stroma (S). Protein samples (10 µ g) were separated by SDS/PAGE, followed by transfer to PVDF membrane, and immunoblotted with antibodies specific for PTOX ( A ). Purity of the fractions was controlled in Arabidopsis and Eutrema by separation and immunoblotting of the samples (5 μg) with antibodies specific for representative polypeptides ( B ). Coomassie brillant blue-stained SDS/PAGE gels of the thylakoid membrane fractions with chlorophyll a / b ratios given below each fraction ( C ). Linearity of the anti-PTOX immunodetection was ensured with respect to the amount of protein per lane. Immunoblot of thylakoid membranes isolated from the control plants of wild-type Eutrema presented ( D ).

Techniques Used: Isolation, SDS Page, Staining, Immunodetection

2) Product Images from "Apoptosis related protein-1 triggers melanoma cell death via interaction with the juxtamembrane region of p75 neurotrophin receptor"

Article Title: Apoptosis related protein-1 triggers melanoma cell death via interaction with the juxtamembrane region of p75 neurotrophin receptor

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2011.01304.x

(A) Dot blots analysis of the interaction of APR-1 protein with TNFR1 human p75NTR, mouse p75NTR, FADD and rat p75NTR. A total of 5 μg of glutathione S -transferase (GST)-TNFR1 (100.0 pmol), TNFR2 (111.0 pmol), human p75NTR (106.4 pmol), mouse p75NTR (106 pmol), FADD (92.6 pmol), TrKA (113.6 pmol), TrKB (113.6 pmol), TrKC (113.6 pmol), Fas 142.8 pmol), death domain (142.8 pmol), APR-1 (100 pmol), rat p75NTR (106 pmol) or GST (192.0 pmol) were diluted in PBS and blotted onto nitrocellulose membrane and subsequently incubated overnight with in vitro transcribed and translated [ 35 S] APR-1 protein. (B) Interaction of APR-1 with P75NTR. The total cell lysates prepared from A375-APR-1 and BLM-APR-1 before and after the induction of APR-1 protein were subjected for either electrophoresis (for the detection of APR-1 and P75NTR) or for co-immunoprecipitation (IP) with either anti-P75NTR antibody or with anti-APR-1 antibody. Western blotting of IP: p75NTR for APR-1 revealed the interaction of APR-1 to P75NTR, whereas Western blotting of IP: APR-1 for P75NTR revealed the interaction of P75NTR to APR-1. β-actin was used as internal control for loading and transfer. (C) Schematic diagram of the extracellular and intracellular domains of p75NTR. Transmembrane domain, JMD and death domain. (D) GST-P75NTR recombinant proteins 1–427aa (106.4 pmol), 1–341aa (135.1 pmol), 1–311aa (147 pmol), 1–274 aa (166 pmol), 275–340aa (694.3) and 341–427aa (526.2 pmol), were separated by SDS-PAGE, and blotted on PVDF membrane and probed with in vitro transcribed and translated [ 35 S] APR-1. The interaction of APR-1 with the P75NTR domains was detected by exposing the membrane to X-ray films. The coomassie-stained gel shows the amount and the position of P75NTR recombinant proteins (left panel). (E) GST-JMD and death domain of P75NTR were separated by SDS-PAGE, and blotted on PVDF membrane and probed with in vitro transcribed and translated [ 35 S] APR-1. The interaction of APR-1 with both domains was detected by exposing the membrane to X-ray films. The coomassie-stained gel shows the amount of both JMD and death domains (left panel). (F) Western blot analysis demonstrates the expression of APR-1 by the addition of Dox to the culture medium of BLM-APR- 1, the knockdown of p75NTR by its specific siRNA and the suppression of APR-1-induced cleavage of PARP by the p75NTR siRNA. β-actin was used as internal control for loading and transfer. (G) Analysis of cell viability by counting using trypan blue staining. Rescue of APR-1-induced reduction of cell viability by the knockdown of p75NTR by siRNA for 24 or 48 hrs. Data are mean of three experiments performed separately.
Figure Legend Snippet: (A) Dot blots analysis of the interaction of APR-1 protein with TNFR1 human p75NTR, mouse p75NTR, FADD and rat p75NTR. A total of 5 μg of glutathione S -transferase (GST)-TNFR1 (100.0 pmol), TNFR2 (111.0 pmol), human p75NTR (106.4 pmol), mouse p75NTR (106 pmol), FADD (92.6 pmol), TrKA (113.6 pmol), TrKB (113.6 pmol), TrKC (113.6 pmol), Fas 142.8 pmol), death domain (142.8 pmol), APR-1 (100 pmol), rat p75NTR (106 pmol) or GST (192.0 pmol) were diluted in PBS and blotted onto nitrocellulose membrane and subsequently incubated overnight with in vitro transcribed and translated [ 35 S] APR-1 protein. (B) Interaction of APR-1 with P75NTR. The total cell lysates prepared from A375-APR-1 and BLM-APR-1 before and after the induction of APR-1 protein were subjected for either electrophoresis (for the detection of APR-1 and P75NTR) or for co-immunoprecipitation (IP) with either anti-P75NTR antibody or with anti-APR-1 antibody. Western blotting of IP: p75NTR for APR-1 revealed the interaction of APR-1 to P75NTR, whereas Western blotting of IP: APR-1 for P75NTR revealed the interaction of P75NTR to APR-1. β-actin was used as internal control for loading and transfer. (C) Schematic diagram of the extracellular and intracellular domains of p75NTR. Transmembrane domain, JMD and death domain. (D) GST-P75NTR recombinant proteins 1–427aa (106.4 pmol), 1–341aa (135.1 pmol), 1–311aa (147 pmol), 1–274 aa (166 pmol), 275–340aa (694.3) and 341–427aa (526.2 pmol), were separated by SDS-PAGE, and blotted on PVDF membrane and probed with in vitro transcribed and translated [ 35 S] APR-1. The interaction of APR-1 with the P75NTR domains was detected by exposing the membrane to X-ray films. The coomassie-stained gel shows the amount and the position of P75NTR recombinant proteins (left panel). (E) GST-JMD and death domain of P75NTR were separated by SDS-PAGE, and blotted on PVDF membrane and probed with in vitro transcribed and translated [ 35 S] APR-1. The interaction of APR-1 with both domains was detected by exposing the membrane to X-ray films. The coomassie-stained gel shows the amount of both JMD and death domains (left panel). (F) Western blot analysis demonstrates the expression of APR-1 by the addition of Dox to the culture medium of BLM-APR- 1, the knockdown of p75NTR by its specific siRNA and the suppression of APR-1-induced cleavage of PARP by the p75NTR siRNA. β-actin was used as internal control for loading and transfer. (G) Analysis of cell viability by counting using trypan blue staining. Rescue of APR-1-induced reduction of cell viability by the knockdown of p75NTR by siRNA for 24 or 48 hrs. Data are mean of three experiments performed separately.

Techniques Used: Incubation, In Vitro, Electrophoresis, Immunoprecipitation, Western Blot, Recombinant, SDS Page, Staining, Expressing

3) Product Images from "A Cleavable Affinity Biotinylating Agent Reveals a Retinoid Binding Role for RPE65"

Article Title: A Cleavable Affinity Biotinylating Agent Reveals a Retinoid Binding Role for RPE65

Journal: Biochemistry

doi: 10.1021/bi034002i

Time-dependent RPE labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after transferring proteins to the PVDF from the 4 to 12% SDS–PAGE gel: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, and 14 kDa); lane 2, RPE control; lane 3, RPE incubated with 1 , at 5 μ M, for 20 s at 4 °C; lane 4, RPE incubated with 1 , at 5 μ M, for 2 min at 4 °C; lane 5, RPE incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 6, RPE incubated with 1 , at 5 μ M, for 30 min at 4 °C; lane 7, RPE incubated with 1 , at 5 μ M, for 1 h at 4 °C; and lane 8, RPE incubated with 1 , at 5 μ M, for 3 h at 4 °C. (B) Time-dependent RPE labeling. The biotin signal is represented by a plot of volume vs time.
Figure Legend Snippet: Time-dependent RPE labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after transferring proteins to the PVDF from the 4 to 12% SDS–PAGE gel: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, and 14 kDa); lane 2, RPE control; lane 3, RPE incubated with 1 , at 5 μ M, for 20 s at 4 °C; lane 4, RPE incubated with 1 , at 5 μ M, for 2 min at 4 °C; lane 5, RPE incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 6, RPE incubated with 1 , at 5 μ M, for 30 min at 4 °C; lane 7, RPE incubated with 1 , at 5 μ M, for 1 h at 4 °C; and lane 8, RPE incubated with 1 , at 5 μ M, for 3 h at 4 °C. (B) Time-dependent RPE labeling. The biotin signal is represented by a plot of volume vs time.

Techniques Used: Labeling, Avidin-Biotin Assay, Transferring, SDS Page, Incubation

2D SDS–PAGE analysis of labeled RPE. (A) RPE proteome in 2D electrophoresis. Proteins were separated by isoelectric focusing (first dimension) and SDS–PAGE (second dimension). An immobilized pH gradient strip (pH 3 to 10, 13 cm) for IEF and a gradient gel (4 to 20%, 16 cm × 18 cm) for SDS–PAGE were used. Proteins were visualized by silver staining. (B) Biotin detection of labeled proteins. RPE was incubated with 1 , at 10 μ M, for 1 h at 4 °C. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL. Biotinylated molecular mass markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa) were loaded in the right-most lane. (C) Unlabeled control RPE. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL.
Figure Legend Snippet: 2D SDS–PAGE analysis of labeled RPE. (A) RPE proteome in 2D electrophoresis. Proteins were separated by isoelectric focusing (first dimension) and SDS–PAGE (second dimension). An immobilized pH gradient strip (pH 3 to 10, 13 cm) for IEF and a gradient gel (4 to 20%, 16 cm × 18 cm) for SDS–PAGE were used. Proteins were visualized by silver staining. (B) Biotin detection of labeled proteins. RPE was incubated with 1 , at 10 μ M, for 1 h at 4 °C. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL. Biotinylated molecular mass markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa) were loaded in the right-most lane. (C) Unlabeled control RPE. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL.

Techniques Used: SDS Page, Labeling, Two-Dimensional Gel Electrophoresis, Stripping Membranes, Electrofocusing, Silver Staining, Incubation, Avidin-Biotin Assay

Competition analysis of RBPs by preblocking and labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after SDS–PAGE on a 4 to 20% gradient gel. Proteins were transferred to a PVDF membrane: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa); lane 2, RPE preblocked with 55 mM iodoacetamide for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 3, RPE preblocked with 1 mM retinol for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 4, RPE preincubated with 1 mM retinyl acetate for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 5, RPE preincubated with 1 mM oleyl acetate and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 6, RPE preincubated with RBA (90 μ M, 1 h) and then 1 , at 5 μ M, for 10 min at 4 °C; lane 7, RPE labeled with 1 , at 5 μ M, for 10 min at 4 °C; lane 8, control RPE; and lane 9, prestained molecular mass markers (177, 114, 81, 64, 50, 37, 26, 20, 15, and 8 kDa). (B) Relative intensity of RPE labeling compared to the control. The biotin signal is represented by volume in the graph.
Figure Legend Snippet: Competition analysis of RBPs by preblocking and labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after SDS–PAGE on a 4 to 20% gradient gel. Proteins were transferred to a PVDF membrane: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa); lane 2, RPE preblocked with 55 mM iodoacetamide for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 3, RPE preblocked with 1 mM retinol for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 4, RPE preincubated with 1 mM retinyl acetate for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 5, RPE preincubated with 1 mM oleyl acetate and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 6, RPE preincubated with RBA (90 μ M, 1 h) and then 1 , at 5 μ M, for 10 min at 4 °C; lane 7, RPE labeled with 1 , at 5 μ M, for 10 min at 4 °C; lane 8, control RPE; and lane 9, prestained molecular mass markers (177, 114, 81, 64, 50, 37, 26, 20, 15, and 8 kDa). (B) Relative intensity of RPE labeling compared to the control. The biotin signal is represented by volume in the graph.

Techniques Used: Labeling, Avidin-Biotin Assay, SDS Page, Incubation

Retinoid affinity biotinylation of RPE. (A) SDS–PAGE gradient gel (4 to 20%). Proteins were visualized by Coomassie blue staining: lane 1 , RPE labeled with 1 , 10 μ M, 1 h at 4 °C; lane 2, RPE labeled with 1 , 100 μ M, 1 h at 4 °C; and lane 3, control RPE. (B) Biotin detection of labeled proteins. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL. Lanes are the same as those for panel A. (C) LRAT Western blot. Proteins were transferred to a PVDF membrane, and LRAT was visualized by anti-LRAT antibody/anti-rabbit Ig-HRP/ECL. Lanes are the same as those for panel A. (D) RPE65 Western blot. Proteins were transferred to a PVDF membrane, and RPE65 was visualized by anti-RPE65 antibody/anti-rabbit Ig-HRP/ECL: lane 1, RPE labeled with 1 , 50 μ M, 1 h at 4 °C; and lane 2, control RPE.
Figure Legend Snippet: Retinoid affinity biotinylation of RPE. (A) SDS–PAGE gradient gel (4 to 20%). Proteins were visualized by Coomassie blue staining: lane 1 , RPE labeled with 1 , 10 μ M, 1 h at 4 °C; lane 2, RPE labeled with 1 , 100 μ M, 1 h at 4 °C; and lane 3, control RPE. (B) Biotin detection of labeled proteins. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL. Lanes are the same as those for panel A. (C) LRAT Western blot. Proteins were transferred to a PVDF membrane, and LRAT was visualized by anti-LRAT antibody/anti-rabbit Ig-HRP/ECL. Lanes are the same as those for panel A. (D) RPE65 Western blot. Proteins were transferred to a PVDF membrane, and RPE65 was visualized by anti-RPE65 antibody/anti-rabbit Ig-HRP/ECL: lane 1, RPE labeled with 1 , 50 μ M, 1 h at 4 °C; and lane 2, control RPE.

Techniques Used: SDS Page, Staining, Labeling, Avidin-Biotin Assay, Western Blot

4) Product Images from "A Cleavable Affinity Biotinylating Agent Reveals a Retinoid Binding Role for RPE65 "

Article Title: A Cleavable Affinity Biotinylating Agent Reveals a Retinoid Binding Role for RPE65

Journal: Biochemistry

doi: 10.1021/bi034002i

Time-dependent RPE labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after transferring proteins to the PVDF from the 4 to 12% SDS–PAGE gel: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, and 14 kDa); lane 2, RPE control; lane 3, RPE incubated with 1 , at 5 μ M, for 20 s at 4 °C; lane 4, RPE incubated with 1 , at 5 μ M, for 2 min at 4 °C; lane 5, RPE incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 6, RPE incubated with 1 , at 5 μ M, for 30 min at 4 °C; lane 7, RPE incubated with 1 , at 5 μ M, for 1 h at 4 °C; and lane 8, RPE incubated with 1 , at 5 μ M, for 3 h at 4 °C. (B) Time-dependent RPE labeling. The biotin signal is represented by a plot of volume vs time.
Figure Legend Snippet: Time-dependent RPE labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after transferring proteins to the PVDF from the 4 to 12% SDS–PAGE gel: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, and 14 kDa); lane 2, RPE control; lane 3, RPE incubated with 1 , at 5 μ M, for 20 s at 4 °C; lane 4, RPE incubated with 1 , at 5 μ M, for 2 min at 4 °C; lane 5, RPE incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 6, RPE incubated with 1 , at 5 μ M, for 30 min at 4 °C; lane 7, RPE incubated with 1 , at 5 μ M, for 1 h at 4 °C; and lane 8, RPE incubated with 1 , at 5 μ M, for 3 h at 4 °C. (B) Time-dependent RPE labeling. The biotin signal is represented by a plot of volume vs time.

Techniques Used: Labeling, Avidin-Biotin Assay, Transferring, SDS Page, Incubation

2D SDS–PAGE analysis of labeled RPE. (A) RPE proteome in 2D electrophoresis. Proteins were separated by isoelectric focusing (first dimension) and SDS–PAGE (second dimension). An immobilized pH gradient strip (pH 3 to 10, 13 cm) for IEF and a gradient gel (4 to 20%, 16 cm × 18 cm) for SDS–PAGE were used. Proteins were visualized by silver staining. (B) Biotin detection of labeled proteins. RPE was incubated with 1 , at 10 μ M, for 1 h at 4 °C. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL. Biotinylated molecular mass markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa) were loaded in the right-most lane. (C) Unlabeled control RPE. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL.
Figure Legend Snippet: 2D SDS–PAGE analysis of labeled RPE. (A) RPE proteome in 2D electrophoresis. Proteins were separated by isoelectric focusing (first dimension) and SDS–PAGE (second dimension). An immobilized pH gradient strip (pH 3 to 10, 13 cm) for IEF and a gradient gel (4 to 20%, 16 cm × 18 cm) for SDS–PAGE were used. Proteins were visualized by silver staining. (B) Biotin detection of labeled proteins. RPE was incubated with 1 , at 10 μ M, for 1 h at 4 °C. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL. Biotinylated molecular mass markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa) were loaded in the right-most lane. (C) Unlabeled control RPE. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL.

Techniques Used: SDS Page, Labeling, Two-Dimensional Gel Electrophoresis, Stripping Membranes, Electrofocusing, Silver Staining, Incubation, Avidin-Biotin Assay

Competition analysis of RBPs by preblocking and labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after SDS–PAGE on a 4 to 20% gradient gel. Proteins were transferred to a PVDF membrane: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa); lane 2, RPE preblocked with 55 mM iodoacetamide for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 3, RPE preblocked with 1 mM retinol for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 4, RPE preincubated with 1 mM retinyl acetate for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 5, RPE preincubated with 1 mM oleyl acetate and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 6, RPE preincubated with RBA (90 μ M, 1 h) and then 1 , at 5 μ M, for 10 min at 4 °C; lane 7, RPE labeled with 1 , at 5 μ M, for 10 min at 4 °C; lane 8, control RPE; and lane 9, prestained molecular mass markers (177, 114, 81, 64, 50, 37, 26, 20, 15, and 8 kDa). (B) Relative intensity of RPE labeling compared to the control. The biotin signal is represented by volume in the graph.
Figure Legend Snippet: Competition analysis of RBPs by preblocking and labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after SDS–PAGE on a 4 to 20% gradient gel. Proteins were transferred to a PVDF membrane: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa); lane 2, RPE preblocked with 55 mM iodoacetamide for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 3, RPE preblocked with 1 mM retinol for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 4, RPE preincubated with 1 mM retinyl acetate for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 5, RPE preincubated with 1 mM oleyl acetate and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 6, RPE preincubated with RBA (90 μ M, 1 h) and then 1 , at 5 μ M, for 10 min at 4 °C; lane 7, RPE labeled with 1 , at 5 μ M, for 10 min at 4 °C; lane 8, control RPE; and lane 9, prestained molecular mass markers (177, 114, 81, 64, 50, 37, 26, 20, 15, and 8 kDa). (B) Relative intensity of RPE labeling compared to the control. The biotin signal is represented by volume in the graph.

Techniques Used: Labeling, Avidin-Biotin Assay, SDS Page, Incubation

Retinoid affinity biotinylation of RPE. (A) SDS–PAGE gradient gel (4 to 20%). Proteins were visualized by Coomassie blue staining: lane 1 , RPE labeled with 1 , 10 μ M, 1 h at 4 °C; lane 2, RPE labeled with 1 , 100 μ M, 1 h at 4 °C; and lane 3, control RPE. (B) Biotin detection of labeled proteins. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL. Lanes are the same as those for panel A. (C) LRAT Western blot. Proteins were transferred to a PVDF membrane, and LRAT was visualized by anti-LRAT antibody/anti-rabbit Ig-HRP/ECL. Lanes are the same as those for panel A. (D) RPE65 Western blot. Proteins were transferred to a PVDF membrane, and RPE65 was visualized by anti-RPE65 antibody/anti-rabbit Ig-HRP/ECL: lane 1, RPE labeled with 1 , 50 μ M, 1 h at 4 °C; and lane 2, control RPE.
Figure Legend Snippet: Retinoid affinity biotinylation of RPE. (A) SDS–PAGE gradient gel (4 to 20%). Proteins were visualized by Coomassie blue staining: lane 1 , RPE labeled with 1 , 10 μ M, 1 h at 4 °C; lane 2, RPE labeled with 1 , 100 μ M, 1 h at 4 °C; and lane 3, control RPE. (B) Biotin detection of labeled proteins. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL. Lanes are the same as those for panel A. (C) LRAT Western blot. Proteins were transferred to a PVDF membrane, and LRAT was visualized by anti-LRAT antibody/anti-rabbit Ig-HRP/ECL. Lanes are the same as those for panel A. (D) RPE65 Western blot. Proteins were transferred to a PVDF membrane, and RPE65 was visualized by anti-RPE65 antibody/anti-rabbit Ig-HRP/ECL: lane 1, RPE labeled with 1 , 50 μ M, 1 h at 4 °C; and lane 2, control RPE.

Techniques Used: SDS Page, Staining, Labeling, Avidin-Biotin Assay, Western Blot

5) Product Images from "Analysis of Toll-Like Receptors in Human Milk: Detection of Membrane-Bound and Soluble Forms"

Article Title: Analysis of Toll-Like Receptors in Human Milk: Detection of Membrane-Bound and Soluble Forms

Journal: Journal of Immunology Research

doi: 10.1155/2019/4078671

Representative image of proteins from MFGM (a) and skimmed milk (b) fractions of colostrum (0) and/or mature milk (2) after SDS-PAGE separation. The name of bands analyzed by mass spectrometry is reported beside each lane. Bands labelled with “B” were digested from PVDF blots, while “G” bands were digested from polyacrylamide gels. S1 was digested from polyacrylamide gels, and S2, S3, S4, S5, S6, S7, S8, S9, and S10 were digested from PVDF blots.
Figure Legend Snippet: Representative image of proteins from MFGM (a) and skimmed milk (b) fractions of colostrum (0) and/or mature milk (2) after SDS-PAGE separation. The name of bands analyzed by mass spectrometry is reported beside each lane. Bands labelled with “B” were digested from PVDF blots, while “G” bands were digested from polyacrylamide gels. S1 was digested from polyacrylamide gels, and S2, S3, S4, S5, S6, S7, S8, S9, and S10 were digested from PVDF blots.

Techniques Used: SDS Page, Mass Spectrometry

6) Product Images from "Expression and functional properties of antibodies to tissue inhibitors of metalloproteinases (TIMPs) in rheumatoid arthritis"

Article Title: Expression and functional properties of antibodies to tissue inhibitors of metalloproteinases (TIMPs) in rheumatoid arthritis

Journal: Arthritis Research & Therapy

doi: 10.1186/ar1771

Western blot analysis of anti-TIMP-2 antibodies. Lysates of THP-1 (a human monocytic cell line) and H9 (a human T-cell lymphoma) were separated in 18% Tris-glycine gel, transferred into a polyvinylidene fluoride membrane, and blotted with immunoglobulin G (IgG) fractions from a patient with rheumatoid arthritis having high levels of anti-TIMP-2 antibodies detected by ELISA. The IgG fraction visualized a band of molecular weight 22 kDa, corresponding to TIMP-2. TIMP, tissue inhibitor of metalloproteinases.
Figure Legend Snippet: Western blot analysis of anti-TIMP-2 antibodies. Lysates of THP-1 (a human monocytic cell line) and H9 (a human T-cell lymphoma) were separated in 18% Tris-glycine gel, transferred into a polyvinylidene fluoride membrane, and blotted with immunoglobulin G (IgG) fractions from a patient with rheumatoid arthritis having high levels of anti-TIMP-2 antibodies detected by ELISA. The IgG fraction visualized a band of molecular weight 22 kDa, corresponding to TIMP-2. TIMP, tissue inhibitor of metalloproteinases.

Techniques Used: Western Blot, Enzyme-linked Immunosorbent Assay, Molecular Weight

7) Product Images from "Gene transduction in mammalian cells using Bombyx mori nucleopolyhedrovirus assisted by glycoprotein 64 of Autographa californica multiple nucleopolyhedrovirus"

Article Title: Gene transduction in mammalian cells using Bombyx mori nucleopolyhedrovirus assisted by glycoprotein 64 of Autographa californica multiple nucleopolyhedrovirus

Journal: Scientific Reports

doi: 10.1038/srep32283

Western blot of GP64 from each baculovirus. Each virus was propagated on Bm5 (BmNPVΔbgp/AcGP64/EGFP and BmNPVΔbgp/BmGP64-EGFP) or Sf-9 (BacMam 2.0) cells and partially purified. Subsequently, 1 × 10 8 or 1 × 10 7 PFU of each virus was separated by SDS-PAGE, transferred to a PVDF membrane, and subjected to western blot analysis using rabbit anti-BmNPV GP64 polyclonal antibody. Lane 1: BmNPVΔbgp/AcGP64/EGFP, Lane 2: BmNPVΔbgp/BmGP64-EGFP, Lane 3: BacMam 2.0. Arrows indicate expressed AcGP64 or BmGP64.
Figure Legend Snippet: Western blot of GP64 from each baculovirus. Each virus was propagated on Bm5 (BmNPVΔbgp/AcGP64/EGFP and BmNPVΔbgp/BmGP64-EGFP) or Sf-9 (BacMam 2.0) cells and partially purified. Subsequently, 1 × 10 8 or 1 × 10 7 PFU of each virus was separated by SDS-PAGE, transferred to a PVDF membrane, and subjected to western blot analysis using rabbit anti-BmNPV GP64 polyclonal antibody. Lane 1: BmNPVΔbgp/AcGP64/EGFP, Lane 2: BmNPVΔbgp/BmGP64-EGFP, Lane 3: BacMam 2.0. Arrows indicate expressed AcGP64 or BmGP64.

Techniques Used: Western Blot, Purification, SDS Page

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Nucleic Acid Electrophoresis:

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Purification:

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Electrophoresis:

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Incubation:

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Polyacrylamide Gel Electrophoresis:

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Western Blot:

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SDS Page:

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    Bio-Rad pvdf membrane
    Relative Accumulation of MatR and Various Mitochondrial Proteins during Arabidopsis Seed Germination. (A) ). Detection was performed by chemiluminescence with the Image Quant LAS4000 mini analyzer (GE Healthcare). The intensities of protein signals in (A) and (B) ). (B) <t>-PAGE</t> analysis of the respiratory chain complexes during seed germination in Arabidopsis. Crude membrane fractions obtained from dry seeds, imbibed seeds, and mature Arabidopsis seedlings were solubilized with DDM ( n ). For immunodetections, the proteins were transferred from the native gels onto a <t>PVDF</t> membrane (Bio-Rad) in cathode buffer for 15 h at 40 mA, using the Bio-Rad mini transblot cell. The membranes were distained with ethanol before probing with specific antibodies, as indicated below each blot. Arrows indicate the native respiratory complexes, CI (∼1000 kD), CIII (dimer, ∼500 kD), CIV (∼220 kD), and CV (∼600 kD), in Arabidopsis mitochondria. Please note, in (B) , the original COX2 blot has been modified, i.e., the lane corresponding of the mature leaves (M) was cut from the right side of the blot and pasted to the other side (marked with dotted line). No other changes have been made to the original figure.
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    Relative Accumulation of MatR and Various Mitochondrial Proteins during Arabidopsis Seed Germination. (A) ). Detection was performed by chemiluminescence with the Image Quant LAS4000 mini analyzer (GE Healthcare). The intensities of protein signals in (A) and (B) ). (B) -PAGE analysis of the respiratory chain complexes during seed germination in Arabidopsis. Crude membrane fractions obtained from dry seeds, imbibed seeds, and mature Arabidopsis seedlings were solubilized with DDM ( n ). For immunodetections, the proteins were transferred from the native gels onto a PVDF membrane (Bio-Rad) in cathode buffer for 15 h at 40 mA, using the Bio-Rad mini transblot cell. The membranes were distained with ethanol before probing with specific antibodies, as indicated below each blot. Arrows indicate the native respiratory complexes, CI (∼1000 kD), CIII (dimer, ∼500 kD), CIV (∼220 kD), and CV (∼600 kD), in Arabidopsis mitochondria. Please note, in (B) , the original COX2 blot has been modified, i.e., the lane corresponding of the mature leaves (M) was cut from the right side of the blot and pasted to the other side (marked with dotted line). No other changes have been made to the original figure.

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    Figure Lengend Snippet: Relative Accumulation of MatR and Various Mitochondrial Proteins during Arabidopsis Seed Germination. (A) ). Detection was performed by chemiluminescence with the Image Quant LAS4000 mini analyzer (GE Healthcare). The intensities of protein signals in (A) and (B) ). (B) -PAGE analysis of the respiratory chain complexes during seed germination in Arabidopsis. Crude membrane fractions obtained from dry seeds, imbibed seeds, and mature Arabidopsis seedlings were solubilized with DDM ( n ). For immunodetections, the proteins were transferred from the native gels onto a PVDF membrane (Bio-Rad) in cathode buffer for 15 h at 40 mA, using the Bio-Rad mini transblot cell. The membranes were distained with ethanol before probing with specific antibodies, as indicated below each blot. Arrows indicate the native respiratory complexes, CI (∼1000 kD), CIII (dimer, ∼500 kD), CIV (∼220 kD), and CV (∼600 kD), in Arabidopsis mitochondria. Please note, in (B) , the original COX2 blot has been modified, i.e., the lane corresponding of the mature leaves (M) was cut from the right side of the blot and pasted to the other side (marked with dotted line). No other changes have been made to the original figure.

    Article Snippet: For nondenaturing-PAGE-protein gel blotting, the gel was transferred to a PVDF membrane (Bio-Rad) in Cathode buffer (50 mM Tricine and 15 mM Bis-Tris-HCl, pH 7.0) for 16 h at 4°C (constant current of 40 mA).

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    Figure Lengend Snippet: RBMY antibody confirmation by using Western-blot analysis of recombinant RBMY. Two concentrations (100 and 10 ng) of each recombinant protein were loaded on SDS-PAGE and after separation were transferred to PVDF membrane. Blots were exposed to X-ray films

    Article Snippet: Total protein extract (40 µg) from each sample was separated on 12% SDS-polyacrylamide gels for 120 min at 100 V and transferred to PVDF membrane (Bio-Rad, USA) by a wet transfer system (Bio-Rad, USA) at 20 V overnight.

    Techniques: Western Blot, Recombinant, SDS Page

    Compounds 33 and 38 bind Aβ and reduce AβO formation, but have no effect on Aβ production. (A) Representative western blot. Cells were treated were lysed, and proteins were separated by SDS-PAGE. After transfer to a PVDF membrane, blots were probed with the 6E10 antibody. (B) Quantification of total Aβ oligomers from western blotting. Error bars represent SEM. (n=6; ** p

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    Figure Lengend Snippet: Compounds 33 and 38 bind Aβ and reduce AβO formation, but have no effect on Aβ production. (A) Representative western blot. Cells were treated were lysed, and proteins were separated by SDS-PAGE. After transfer to a PVDF membrane, blots were probed with the 6E10 antibody. (B) Quantification of total Aβ oligomers from western blotting. Error bars represent SEM. (n=6; ** p

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    Techniques: Western Blot, SDS Page

    Relative accumulation of organellar proteins in wild-type and mterf22 plants. (A) Immunoblot analyses of wild-type plants and mterf22-1 mutant line. For the quantification of the relative abundances of organellar proteins in mterf22 plants, different amounts of total mitochondrial proteins extracted from wild-type plants were loaded and separated by SDS-PAGE. The blots were probed with polyclonal antibodies raised to different organellar proteins, as indicated in each panel. Detection was carried out by chemiluminescence assays after incubation with HRP-conjugated secondary antibody. (B) BN-PAGE of crude mitochondria preparations was performed according to the method described in [ 57 ]. Crude mitochondria preparations, obtained from 3-week-old Arabidopsis seedlings, were solubilized with DDM [1.5% (w/v)] and the organellar complexes were resolved by BN-PAGE. For immunodetection, proteins were transferred from the native gels onto a PVDF membrane and were probed with specific antibodies ( S2 Table ), as indicated below each blot. Arrows indicate to the native complexes I (~1,000 kDa), III (dimer, ~500 kDa), IV (~220 kDa) and V (~600 kDa). The asterisk in the CA2 panel indicates to the presence of a 700 ~ 800 kDa band, which may corresponds to a complex I assembly intermediate. Hybridization signals were analyzed by chemiluminescence assays after incubation with HRP-conjugated secondary antibody. The intensities of protein signals in panels ‘A’ and ‘B’ using ImageJ software [ 90 ].

    Journal: PLoS ONE

    Article Title: Control of organelle gene expression by the mitochondrial transcription termination factor mTERF22 in Arabidopsis thaliana plants

    doi: 10.1371/journal.pone.0201631

    Figure Lengend Snippet: Relative accumulation of organellar proteins in wild-type and mterf22 plants. (A) Immunoblot analyses of wild-type plants and mterf22-1 mutant line. For the quantification of the relative abundances of organellar proteins in mterf22 plants, different amounts of total mitochondrial proteins extracted from wild-type plants were loaded and separated by SDS-PAGE. The blots were probed with polyclonal antibodies raised to different organellar proteins, as indicated in each panel. Detection was carried out by chemiluminescence assays after incubation with HRP-conjugated secondary antibody. (B) BN-PAGE of crude mitochondria preparations was performed according to the method described in [ 57 ]. Crude mitochondria preparations, obtained from 3-week-old Arabidopsis seedlings, were solubilized with DDM [1.5% (w/v)] and the organellar complexes were resolved by BN-PAGE. For immunodetection, proteins were transferred from the native gels onto a PVDF membrane and were probed with specific antibodies ( S2 Table ), as indicated below each blot. Arrows indicate to the native complexes I (~1,000 kDa), III (dimer, ~500 kDa), IV (~220 kDa) and V (~600 kDa). The asterisk in the CA2 panel indicates to the presence of a 700 ~ 800 kDa band, which may corresponds to a complex I assembly intermediate. Hybridization signals were analyzed by chemiluminescence assays after incubation with HRP-conjugated secondary antibody. The intensities of protein signals in panels ‘A’ and ‘B’ using ImageJ software [ 90 ].

    Article Snippet: For immunoblotting of non-denaturing PAGE, the proteins were transferred from the gel onto a PVDF membrane (Bio-Rad) in Cathode buffer (50 mM Tricine, 15 mM Bis-Tris-HCl, pH 7.0) for 16 h at 4°C at constant current of 40 mA.

    Techniques: Mutagenesis, SDS Page, Incubation, Polyacrylamide Gel Electrophoresis, Immunodetection, Hybridization, Software