Journal: Cell

Article Title: A membrane-associated MHC-I inhibitory axis for cancer immune evasion

doi: 10.1016/j.cell.2023.07.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For intracellular staining, cells were permeabilized with 0.1% saponin in PBS + 2% BSA containing Fc blocker for 30 min at room temperature, then stained overnight at 4 °C with anti-mouse EEA1 antibody (Thermo Fisher Scientific # MA5–31575, dilution 1:100), or APC anti-mouse CD107a (LAMP1) (Miltenyi #130–111-505, dilution 1:50), or APC anti-mouse CD71 (Miltenyi #130–119-133, dilution 1:50).

Techniques: Control, Purification, Blocking Assay, Virus, Recombinant, Protease Inhibitor, Transfection, Immunoprecipitation, Activation Assay, Magnetic Beads, Reverse Transcription, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Double Knockout, shRNA, Real-time Polymerase Chain Reaction, Genome Wide, Plasmid Preparation, Software, Flow Cytometry

Journal:

Article Title: The RCP-Rab11 Complex Regulates Endocytic Protein Sorting D⃞

doi: 10.1091/mbc.E03-12-0918

Figure Lengend Snippet: Subcellular localization of RCP. Tf-TxR (A–C, red) was bound to HeLa cells for 1 h at 4°C and then incubated at 37°C for 5 min (A) or 30 min (B and C). Cells were then fixed and stained with anti-RCP antibodies (A and C, green). In D, fixed HeLa cells were costained with anti-RCP (green) and anti-EEA1 (red) antibodies. In E and F, HeLa cells transfected with RCP-YFP and CFP-Rab11A were incubated with Tf-TxR for 1 h at 4°C and then incubated at 37°C for 30 min. Colocalization between Tf-TxR (E and F, red) and RCP–YFP/CFP–Rab11 complex was then determined using FRET (F, green). In G–J, HeLa cells were treated with either nocodazole (I and J) or brefeldin A (G and H), fixed, and stained with anti-RCP (H and J, green) or anti-TfR (G–J, red) antibodies. The yellow signal in C, F, H, and J, represents overlap. Bars, 2 μm.

Article Snippet: Mouse anti-transferrin receptor-phycoerythrin (TfR-PE) was purchased from BD Biosciences.

Techniques: Incubation, Staining, Transfection

Journal:

Article Title: The RCP-Rab11 Complex Regulates Endocytic Protein Sorting D⃞

doi: 10.1091/mbc.E03-12-0918

Figure Lengend Snippet: RCP recruitment to endosomes is dependent on binding to Rab11. HeLa cells were incubated in the presence (A–C, E–F) or absence (D) of digitonin. Cells were then washed and incubated with either GST (C) or GST-RCP-F1 (A and B, D–F) in the presence (E and F) or absence (A–D) of soluble recombinant Rab11. Cells were fixed and stained with anti-GST (B and F, green; and C and D, gray) and anti-TfR (A and B and E and F, red) antibodies. Yellow in B and F represents the degree of overlap. Bars, 3 μm.

Article Snippet: Mouse anti-transferrin receptor-phycoerythrin (TfR-PE) was purchased from BD Biosciences.

Techniques: Binding Assay, Incubation, Recombinant, Staining

Journal:

Article Title: The RCP-Rab11 Complex Regulates Endocytic Protein Sorting D⃞

doi: 10.1091/mbc.E03-12-0918

Figure Lengend Snippet: Overexpression of RCP-F1 inhibits Tf recycling and uptake. (A and B) HeLa cells expressing either GFP or GFP-RCP-F1 were incubated at 4°C for 30 min with 2 μg/ml anti-TfR-PE antibody. The amount of plasma membrane bound anti-TfR-PE antibody after this incubation was quantitated by FACS and is shown in A. Cells were then incubated at 37°C for varying amounts of time in the continuous presence of 2 μg/ml anti-TfR-PE antibody. The amount of internalized anti-TfR-PE antibody was quantitated by FACS analysis and is shown in B. The data shown are the means of at least three independent experiments. To better compare the rates of uptake, the plasma membrane-bound anti-TfR-PE after 4°C incubation was subtracted from the data in A. (C) HeLa cells expressing either GFP or GFP-RCP-F1 were incubated at 4°C for 30 min with 20 μg/ml Tf-Alexa647. Cells were then incubated at 37°C for varying amounts of time in the continuous presence of 20 μg/ml Tf-Alexa647. The amount of internalized Tf-Alexa647 was quantitated by FACS analysis. The data shown are the means of at least three independent experiments. (D) HeLa cells expressing either GFP or GFP-RCP-F1 were incubated for 30 min with Tf-Alexa647 at 4°C. Cells were then incubated for an additional 20 min at 37°C in the presence of Tf-Alexa647. The amounts of internalized Tf-Alexa647 after incubation at 37°C are shown in the inset. Cells were then washed and incubated at 37°C for varying amounts of time in the presence of unlabeled Tf. The Tf-Alexa647 remaining in the cells was quantitated using FACS analysis and expressed as a percentage of total endocytosed (at time 0) Tf-Alexa647.

Article Snippet: Mouse anti-transferrin receptor-phycoerythrin (TfR-PE) was purchased from BD Biosciences.

Techniques: Over Expression, Expressing, Incubation

Journal:

Article Title: The RCP-Rab11 Complex Regulates Endocytic Protein Sorting D⃞

doi: 10.1091/mbc.E03-12-0918

Figure Lengend Snippet: Overexpression of GFP-RCP-F1 inhibits TfR recycling from endosomes to the plasma membrane. HeLa cells were mock transfected (A–C) or transfected with GFP-RCP-F1 (D–F). Cells were incubated with Tf-TxR for 1 h at 4°C; washed; incubated at 37°C for 5 min (A and D), 20 min (B and E), and 60 min (C and F); and fixed. The localization and fluorescence intensities of Tf-TxR and GFP-RCP-F1 were then imaged. The colocalization between Tf-TxR and RCP-F1-GFP are the means of five randomly picked cells. In G and H, HeLa cells were transfected with GFP-RCP-F1 (G) and stained with anti-TfR antibodies (H).

Article Snippet: Mouse anti-transferrin receptor-phycoerythrin (TfR-PE) was purchased from BD Biosciences.

Techniques: Over Expression, Transfection, Incubation, Fluorescence, Staining

Journal:

Article Title: The RCP-Rab11 Complex Regulates Endocytic Protein Sorting D⃞

doi: 10.1091/mbc.E03-12-0918

Figure Lengend Snippet: Effect of RCP knock-down on TfR endocytosis, lysosomal degradation and recycling. (A) HeLa cells were either mock transfected or transfected with Rip11 or RCP siRNAs, and Triton X-100 extracts of them were analyzed by Western blotting with anti-RCP, anti-Rip11, anti-Rab11, and anti-γ adaptin antibodies to determine the extent and specificity of the knock-down. (B) HeLa cells were either mock transfected (control) or transfected with Rip11 or RCP siRNAs. After 72 h, cells were incubated for 30 min at 4°C with 20 μg/ml Tf-Alexa647. Cells were then incubated for additional 30 min at 37°C in the presence of Tf-Alexa647. Cells were then washed, fixed in 3% paraformaldehyde, and amounts of internalized Tf-Alexa647 were quantitated by FACS. The data presented are means ± SE of three independent experiments. (C) HeLa cells were either mock transfected or transfected with Rip11 or RCP siRNAs. After 72 h, cells were incubated for 30 min at 4°C with 2 μg/ml anti-TfR-PE antibody. The amount of plasma membrane bound anti-TfR-PE antibody is shown in the inset. Cells were then moved to at 37°C and incubated for varying amounts of time in the presence of 20 μg/ml Tf-Alexa647. Cells were then washed, fixed in 3% paraformaldehyde, and the time course of anti-TfR-PE antibody internalization was quantitated by FACS analysis. (D) HeLa cells were either mock transfected (control) or transfected with Rip11 or RCP siRNAs. After 72 h, cells were incubated for 30 min with Tf-Alexa647 at 4°C followed by additional 20-min incubation at 37°C. Cells were then washed and incubated at 37°C for 20 min in the presence of unlabeled Tf. The amounts of cell associated Tf-Alexa647 was quantitated by FACS. The data presented are means ± SE of three independent experiments.

Article Snippet: Mouse anti-transferrin receptor-phycoerythrin (TfR-PE) was purchased from BD Biosciences.

Techniques: Transfection, Western Blot, Incubation

Journal:

Article Title: The RCP-Rab11 Complex Regulates Endocytic Protein Sorting D⃞

doi: 10.1091/mbc.E03-12-0918

Figure Lengend Snippet: RCP knock-down increases lysosomal degradation of TfR but not EGFR. (A and C) HeLa cells were either mock transfected or transfected with RCP or Rip11 siRNAs. After 74-h incubation, cells were either permeabilized with saponin to measure total TfR (A) or nonpermeabilized to measure cell surface TfR (C), stained with anti-TfR-PE antibodies, and quantitated by FACS analysis or Western blotting (A, inset). (B and D) Quantitation of data from A and C. The presented data are means ± SD of three independent experiments. (E and F) Cells were transfected with RCP siRNA, fixed, and stained with anti-RCP (E) and anti-TfR (F) antibodies. Arrow points to a HeLa cell with down-regulated RCP. Bars, 2 μM. (G and H) HeLa cells were either mock transfected or transfected with RCP siRNAs. After 74-h incubation, cells were either permeabilized to measure total EGFR (G) or nonpermeabilized to measure cell surface EGFR (H). The levels of EGFR were quantitated by FACS analysis.

Article Snippet: Mouse anti-transferrin receptor-phycoerythrin (TfR-PE) was purchased from BD Biosciences.

Techniques: Transfection, Incubation, Staining, Western Blot, Quantitation Assay