Journal: Developmental cell

Article Title: The Rag GTPase regulates the dynamic behavior of TSC downstream of both amino acid and growth factor restriction

doi: 10.1016/j.devcel.2020.08.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: RagA (D8B5) Rabbit mAb , Cell Signaling Technology , Cat# 4357, RRID:AB_10545136.

Techniques: Recombinant, Clinical Proteomics, Electron Microscopy, Control, Plasmid Preparation, Software, Modification, Transfection, Protein Extraction, Blocking Assay, Western Blot

Journal: Molecular Vision

Article Title: Ischemia-reperfusion injury of the retina is linked to necroptosis via the ERK1/2-RIP3 pathway

doi:

Figure Lengend Snippet: Schematic representation of RAGE protein domains. The position of the glycosylation sites, disulfide bonds, and recognition areas for the antibodies used in this experiment are shown. The monoclonal anti-RAGE antibody shown in pink recognizes the extracellular domains of full-length RAGE. The polyclonal anti-RAGE antibody, shown in blue, recognizes the cytoplasmic tail of full-length RAGE.

Article Snippet: To prevent binding of the secondary anti-rat antibody to endogenous rat tissue immunoglobulin (IgG), the frozen sections were preincubated with unconjugated AffiniPure Fab fragments of donkey anti-rat IgG (H+L; Jackson ImmunoResearch Labs, West Grove, PA, Cat# 712–007–003) for 1 h. The sections were incubated overnight with primary antibodies against either RAGE (1:500, Abcam, Ab3611), RAGE (1:50, R&D, Mab1179, MN), poly ADP-ribose polymerase (PARP; 1:50, Abcam, Ab32138), or Brn3a (1:500, Santa Cruz, SC-31984) at 4 °C.

Techniques:

Journal: Molecular Vision

Article Title: Ischemia-reperfusion injury of the retina is linked to necroptosis via the ERK1/2-RIP3 pathway

doi:

Figure Lengend Snippet: Western blots indicating the presence of the RAGE protein in the retina of the IR-injured rat. Six time points are presented on each blot. Every time point contained the experimental group (E) and the sham control group (S). A : The R&D-anti-RAGE antibody detected increased eRAGE accumulation at 12 h post ischemia. B : The peptide competition assay confirmed the specific band reactivity of the R&D-anti-RAGE antibody; lane- a western blot with R&D-anti-RAGE antibody preincubated with blocking peptide; lane- b western blot with a R&D-anti-RAGE antibody not preincubated with peptide. C : Western blots with R&D-anti-RAGE antibody performed under reducing and non-reducing conditions; lane- a non-reducing condition; lane- b reducing condition (β-mercaptoethanol); lane- c reducing condition (β-mercaptoethanol and dithiothreitol (DTT)). D : Abcam-anti RAGE-antibody shows the presence of cRAGE in every post-ischemic time point in the experimental and sham control animals. No significant difference in the accumulation of the cRAGE protein was detected among the examined post-ischemic time points.

Article Snippet: To prevent binding of the secondary anti-rat antibody to endogenous rat tissue immunoglobulin (IgG), the frozen sections were preincubated with unconjugated AffiniPure Fab fragments of donkey anti-rat IgG (H+L; Jackson ImmunoResearch Labs, West Grove, PA, Cat# 712–007–003) for 1 h. The sections were incubated overnight with primary antibodies against either RAGE (1:500, Abcam, Ab3611), RAGE (1:50, R&D, Mab1179, MN), poly ADP-ribose polymerase (PARP; 1:50, Abcam, Ab32138), or Brn3a (1:500, Santa Cruz, SC-31984) at 4 °C.

Techniques: Western Blot, Competitive Binding Assay, Blocking Assay

Journal: Molecular Vision

Article Title: Ischemia-reperfusion injury of the retina is linked to necroptosis via the ERK1/2-RIP3 pathway

doi:

Figure Lengend Snippet: Immunohistochemical staining of the RAGE protein in the rat retina. A : The eRAGE protein is absent in the naïve (wild-type, WT) rat retina. B : The eRAGE protein accumulates in the retinal ganglion layer (RGC) layer of the retina at the 12 h post-ischemic time point. C : The eRAGE protein is not present in the retina at the 1 d post-ischemia-reperfusion period. The R&D anti-RAGE antibody (red) was used. Scale bars=20 μm.

Article Snippet: To prevent binding of the secondary anti-rat antibody to endogenous rat tissue immunoglobulin (IgG), the frozen sections were preincubated with unconjugated AffiniPure Fab fragments of donkey anti-rat IgG (H+L; Jackson ImmunoResearch Labs, West Grove, PA, Cat# 712–007–003) for 1 h. The sections were incubated overnight with primary antibodies against either RAGE (1:500, Abcam, Ab3611), RAGE (1:50, R&D, Mab1179, MN), poly ADP-ribose polymerase (PARP; 1:50, Abcam, Ab32138), or Brn3a (1:500, Santa Cruz, SC-31984) at 4 °C.

Techniques: Immunohistochemical staining, Staining

Journal: Molecular Vision

Article Title: Ischemia-reperfusion injury of the retina is linked to necroptosis via the ERK1/2-RIP3 pathway

doi:

Figure Lengend Snippet: Colocalization of RAGE with Brn3a and PARP in the IR-injured rat retina at the12 h post-ischemia-reperfusion period. A : The transcription factor BRN3a (green) is present in a subset of cells in the retinal ganglion layer (RGC) layer at the12 h post-ischemia-reperfusion period. B : eRAGE (red) is localized to cells in the RGC layer in the ischemia reperfusion (IR)-injured rat retina at 12 h post-IR. C : Double immunolabeling with the eRAGE antibody (red) and the ganglion cell marker, BRN3a (green), at the 12 h reperfusion time shows they are colocalized. D : Necrotic cell marker, poly ADP-ribose polymerase (PARP) (green), is localized to cells in the RGC layer in the IR-injured rat retina at 12 h post-IR. E : eRAGE (red) is localized to cells in the RGC layer in the IR-injured rat retina at the 12 h post-ischemia-reperfusion period. F : Double immunolabeling with the RAGE antibody (red) and PARP at 12 h post-IR shows they are colocalized. Scale bars=20 μm.

Article Snippet: To prevent binding of the secondary anti-rat antibody to endogenous rat tissue immunoglobulin (IgG), the frozen sections were preincubated with unconjugated AffiniPure Fab fragments of donkey anti-rat IgG (H+L; Jackson ImmunoResearch Labs, West Grove, PA, Cat# 712–007–003) for 1 h. The sections were incubated overnight with primary antibodies against either RAGE (1:500, Abcam, Ab3611), RAGE (1:50, R&D, Mab1179, MN), poly ADP-ribose polymerase (PARP; 1:50, Abcam, Ab32138), or Brn3a (1:500, Santa Cruz, SC-31984) at 4 °C.

Techniques: Immunolabeling, Marker

Journal: Molecular Vision

Article Title: Ischemia-reperfusion injury of the retina is linked to necroptosis via the ERK1/2-RIP3 pathway

doi:

Figure Lengend Snippet: Localization of the cRAGE protein in the retina from control, 12 h post-ischemia reperfusion (IR), 1 day post-IR, and 3 days post-IR, using the Abcam-anti-RAGE antibody. The cRAGE protein was detected mainly in the ganglion cell and inner nuclear layers. Scale bars=50 μm.

Article Snippet: To prevent binding of the secondary anti-rat antibody to endogenous rat tissue immunoglobulin (IgG), the frozen sections were preincubated with unconjugated AffiniPure Fab fragments of donkey anti-rat IgG (H+L; Jackson ImmunoResearch Labs, West Grove, PA, Cat# 712–007–003) for 1 h. The sections were incubated overnight with primary antibodies against either RAGE (1:500, Abcam, Ab3611), RAGE (1:50, R&D, Mab1179, MN), poly ADP-ribose polymerase (PARP; 1:50, Abcam, Ab32138), or Brn3a (1:500, Santa Cruz, SC-31984) at 4 °C.

Techniques:

Journal: Molecular Vision

Article Title: Ischemia-reperfusion injury of the retina is linked to necroptosis via the ERK1/2-RIP3 pathway

doi:

Figure Lengend Snippet: Localization of the RAGE protein in the retina using the two anti-RAGE antibodies at 12 h post-ischemia. A : The Abcam-anti-RAGE antibody (green) localized the cRAGE protein in the nucleus only. B : The R&D-anti-RAGE antibody (red) localized the eRAGE protein in the cytoplasm and the nucleus. C : eRAGE and cRAGE colocalized in cells of the retinal ganglion layer (RGC) layer at the 12 h post-ischemic time point. The colocalization shows the nucleus contains cRAGE and eRAGE. Scale bars=20 μm.

Article Snippet: To prevent binding of the secondary anti-rat antibody to endogenous rat tissue immunoglobulin (IgG), the frozen sections were preincubated with unconjugated AffiniPure Fab fragments of donkey anti-rat IgG (H+L; Jackson ImmunoResearch Labs, West Grove, PA, Cat# 712–007–003) for 1 h. The sections were incubated overnight with primary antibodies against either RAGE (1:500, Abcam, Ab3611), RAGE (1:50, R&D, Mab1179, MN), poly ADP-ribose polymerase (PARP; 1:50, Abcam, Ab32138), or Brn3a (1:500, Santa Cruz, SC-31984) at 4 °C.

Techniques: