Journal: Theranostics

Article Title: Combinative treatment of β-elemene and cetuximab is sensitive to KRAS mutant colorectal cancer cells by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation

doi: 10.7150/thno.44705

Figure Lengend Snippet: Combinative treatment of β-elemene and cetuximab was sensitive to KRAS mutant CRC cells. (A) The sensitivity of KRAS mutant and wild-type colorectal cancer cells to cetuximab treatment (25 µg/ml) for 24 h was detected by CCK-8 assay. The mean ± s.d. is shown. ** P < 0.01. (B) The inhibitory effects and cytotoxicity of co-treatment with β-elemene (125 µg/ml) and cetuximab (25 µg/ml) in KRAS mutant CRC cells was determined after the treatment for 24 h. (C) Representative cell morphological changes are detected by light microscopy. Scale bar = 100 μm. (D) Representative results of annexin V-FITC/PI staining and quantitative analysis after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml) for 24 h. The mean ± s.d. is shown. ** P < 0.01. (E) Representative results of cell cycle and quantitative analysis after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml) for 24 h. (F) The colony-formation assay was performed and colony numbers are shown (β-elemene 125 µg/ml, cetuximab 25 µg/ml). The mean ± s.d. is shown. ** P < 0.01.

Article Snippet: Cetuximab (#33657) was purchased from MCE.

Techniques: Mutagenesis, CCK-8 Assay, Light Microscopy, Staining, Colony Assay

Journal: Theranostics

Article Title: Combinative treatment of β-elemene and cetuximab is sensitive to KRAS mutant colorectal cancer cells by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation

doi: 10.7150/thno.44705

Figure Lengend Snippet: The effect of co-treatment with β-elemene and cetuximab on several ferroptotic events in KRAS mutant CRC cells. (A) The effect of cetuximab and β-elemene in combination with other cell death inhibitors on the cell viability of KRAS mutant HCT116 and Lovo cells after the treatment for 24 h. The mean ± s.d. is shown. (B) The cellular ROS level after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml) for 24 h was analyzed by a flow cytometer, ** P < 0.01. (C) Intracellular GSH level in KRAS mutant HCT116 and Lovo cells after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml) for 24 h was detected, ** P < 0.01. (D) Intracellular MDA levels in KRAS mutant HCT116 and Lovo cells after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml) for 24 h was detected, ** P < 0.01.

Article Snippet: Cetuximab (#33657) was purchased from MCE.

Techniques: Mutagenesis, Flow Cytometry

Journal: Theranostics

Article Title: Combinative treatment of β-elemene and cetuximab is sensitive to KRAS mutant colorectal cancer cells by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation

doi: 10.7150/thno.44705

Figure Lengend Snippet: The iron ion level and mitochondria staining were detected. (A) The chelatable iron was determined using the fluorescent indicator Phen Green SK (green) after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml) for 24 h. Scale bar = 100 µm. (B) The Mitochondria morphology was assessed with Mito-Tracker Green after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml) for 24 h. Scale bar = 50 µm.

Article Snippet: Cetuximab (#33657) was purchased from MCE.

Techniques: Staining

Journal: Theranostics

Article Title: Combinative treatment of β-elemene and cetuximab is sensitive to KRAS mutant colorectal cancer cells by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation

doi: 10.7150/thno.44705

Figure Lengend Snippet: The effect of co-treatment with β-elemene and cetuximab on ferroptosis-related proteins in KRAS mutant CRC cells. (A) The expression of positive regulatory proteins for ferroptosis (HO-1 and transferrin) and the negative regulatory proteins for ferroptosis (GPX4, SLC7A11, FTH1, glutaminase, and SLC40A1) were detected by western blotting after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml) for 24 h. (B) HCT116 and Lovo cells were treated with cetuximab (25 µg/ml) and β-elemene (125 µg/ml) with or without ferroptosis inhibitors for 24 h and cell viability was assayed. The mean ± s.d. is shown. ** P < 0.01.

Article Snippet: Cetuximab (#33657) was purchased from MCE.

Techniques: Mutagenesis, Expressing, Western Blot

Journal: Theranostics

Article Title: Combinative treatment of β-elemene and cetuximab is sensitive to KRAS mutant colorectal cancer cells by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation

doi: 10.7150/thno.44705

Figure Lengend Snippet: Combinative treatment of β-elemene and cetuximab suppressed the migration of KRAS mutant CRC cells by inhibiting EMT. (A) Representative results of wound healing after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml, DFO 20 nM) for 24 h. The mean ± s.d. is shown. ** P < 0.01. (B) Transwell invasion assay was performed by the 24-transwell system and quantitative analysis. The pictures were taken 24 h after seeding (original magnification: × 100). The mean ± s.d. is shown. ** P < 0.01. (C) The expression of several key EMT markers Vimentin, E-Cadherin, N-Cadherin, Slug, Snail and MMP-9 were detected after the treatment (β-elemene 125 µg/ml, cetuximab 25 µg/ml) for 24 h by western blotting.

Article Snippet: Cetuximab (#33657) was purchased from MCE.

Techniques: Migration, Mutagenesis, Transwell Invasion Assay, Expressing, Western Blot

Journal: Theranostics

Article Title: Combinative treatment of β-elemene and cetuximab is sensitive to KRAS mutant colorectal cancer cells by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation

doi: 10.7150/thno.44705

Figure Lengend Snippet: The antitumor efficacy of co-treatment with β-elemene and cetuximab in vivo . (A) The scheme of tumor inoculation and systemic injection. (B) Bioluminescent imaging for HCT116-luc orthotopic xenograft colon tumors at different time points post treatment (β-elemene 50 mg/kg, cetuximab 50 mg/kg) and representative image of metastatic lymph nodes. (C) Fold change in average radiance per mouse at experimental endpoint (day 18) was analyzed for each treatment group. Data are expressed as the mean ± s.d. (D) The survival curves of mice in each group were assessed.

Article Snippet: Cetuximab (#33657) was purchased from MCE.

Techniques: In Vivo, Injection, Imaging

Journal: Science Advances

Article Title: TET2 regulates early and late transitions in exhausted CD8 + T cell differentiation and limits CAR T cell function

doi: 10.1126/sciadv.adp9371

Figure Lengend Snippet: ( A ) Left: Schematic of TRAC (top) and TET2 (bottom) loci alongside rAAV6 KI vectors. Right: Sanger sequencing electropherogram confirming integration of TRAC and TET2 KI constructs, underlined with dashed line. ( B ) Example plots of TET2 and TRAC-CAR19 single KI or dual TET2-TRAC-CAR19 KI T cells. ( C ) Example plots of CD3 loss detected by flow in TRAC-CAR19-KI T cells. ( D and E ) Schematic of in vitro ADCC assay (D) to deplete CRISPR-edited TET2-KI T cells. Example plots and data (E) of EGFR expression on TET2-KI T cells alone or in an NK cell coculture ± cetuximab incubation, gated on CD56 − populations, n = 4. ( F ) Cumulative fold expansion of TRAC-CAR19 and TET2-TRAC-CAR19 T cells during restimulation and at day 25, arrows represent addition of irradiated K562-CD19 + target cells, n = 5. ( G ) Proportions of CD4 + versus CD8 + T cells in TRAC-CAR19 and TET2-TRAC-CAR19 T cells after stimulation, n = 7. ( H ) Example plots showing distribution of central (CCR7 + CD45RO + ) and effector (CCR7 − CD45RO + ) memory-associated markers in CD8 + CAR T cell populations after restimulation, with summary after five stimulations, n = 5. ( I ) SPICE plot showing distribution of IR coexpression in CD8 + TRAC-CAR19 and TET2-TRAC-CAR19 T cells after 1 (acute) and 5 (chronic) stimulations, n = 6. ( J ) Data shown as means ± SEM [(F) and (G)] or individual values [(E) and (H)] from independent donors. ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001 by paired t test. Schematics [(A and (D)] created with BioRender.com .

Article Snippet: The following day, TET2 KI T cells were incubated with a cetuximab biosimilar (R&D Systems, no. MAB9577) at a concentration of 2000 ng/ml for 20 min. T cells were then cocultured at a 1:10 ratio with NK cells, with T cell numbers normalized to EGFR + expression.

Techniques: Sequencing, Construct, In Vitro, ADCC Assay, CRISPR, Expressing, Incubation, Irradiation

Journal: PLoS ONE

Article Title: Loss of p120ctn causes EGFR-targeted therapy resistance and failure

doi: 10.1371/journal.pone.0241299

Figure Lengend Snippet: (A) Cell viability assays in EPC1-C, -P, -E, and -PE cells show that EPC1-PE cells are resistant to EGFR therapy-induced death, (n = 8). (B) Cell viability assays show that EPC2-PE cells are resistant to cell death, (n = 4). Vehicle control was DMSO. * denotes p<0.05 for gefitinib treatment comparisons between EPC-E vs. EPC-C and EPC-PE vs. EPC-E. # denotes p<0.05 for erlotinib treatment comparisons between EPC-E vs. EPC-C and EPC-PE vs. EPC-E. ^ denotes p<0.05 for cetuximab treatment comparisons between EPC-E vs. EPC-C and EPC-PE vs. EPC-E.

Article Snippet: For viability testing, cells were treated with 5 μM gefitinib (#076091; Matrix Scientific, Columbia, SC), 10 μM erlotinib (#10483; Cayman Chemical; Ann Arbor, MI), or 10 nM cetuximab (#NBP2-75903; Novus Biologicals; Centennial, CO), with or without BAY 11–7085 (#B3033; ApexBio Technology; Houston, TX) at 2 μM or 3.3 μM for 48 hours.

Techniques: Control

Journal: PLoS ONE

Article Title: Loss of p120ctn causes EGFR-targeted therapy resistance and failure

doi: 10.1371/journal.pone.0241299

Figure Lengend Snippet: (A) A dose response curve for BAY 11–7085 using EPC1 cells demonstrates no significant cell death in any cell line at 2 uM. (B) Western blot analysis demonstrates a decrease in pNFkB expression when cells are treated with 2 uM BAY 11–7085. (C) Cell viability assays demonstrate that treatment of EPC1 cells with 2 uM BAY 11–7085 in combination with either gefitinib, erlotinib, or cetuximab results in a partial reduction in cell viability in EPC1-PE cells (n = 3). Vehicle refers to cells treated with 2 uM BAY 11–7085 and DMSO (as the EGFR diluent). * denotes p<0.05 for gefitinib treatment comparisons between EPC-E vs. EPC-C and EPC-PE vs. EPC-E. # denotes p<0.05 for erlotinib treatment comparisons between EPC-E vs. EPC-C and EPC-PE vs. EPC-E. ^ denotes p<0.05 for cetuximab treatment comparisons between EPC-E vs. EPC-C and EPC-PE vs. EPC-E.

Article Snippet: For viability testing, cells were treated with 5 μM gefitinib (#076091; Matrix Scientific, Columbia, SC), 10 μM erlotinib (#10483; Cayman Chemical; Ann Arbor, MI), or 10 nM cetuximab (#NBP2-75903; Novus Biologicals; Centennial, CO), with or without BAY 11–7085 (#B3033; ApexBio Technology; Houston, TX) at 2 μM or 3.3 μM for 48 hours.

Techniques: Western Blot, Expressing

Journal: PLOS One

Article Title: Microfluidic isolation and release of live disseminated breast tumor cells in bone marrow

doi: 10.1371/journal.pone.0319392

Figure Lengend Snippet: (A) Genes were all highly expressed in two cancer cell lines and barely expressed in BM samples. Specifically, there were no detectable levels of EGFR and EpCAM in both BM samples. MDA: MDA-MB-231; MCF7: MCF-7; BM1, BM2: bone marrow cells from healthy donors #1 and #2; BM1+1, +5, +50: 1, 5, 50 MDA-MB-231 cells were spiked into 1 million BM cells from healthy donor #1. (B) EGFR was highly expressed in MDA-MB-231 cells, an observation that aligned with the literature. Nucleated human BM cells were used as control. Streptavidin-PE was used to label biotinylated antibodies.

Article Snippet: Biotinylated antibody against epithelial cell adhesion molecule (anti-EpCAM) (eBioscience, Carlsbad, CA, USA) and biotinylated antibody against epidermal growth factor receptor (anti-EGFR, Research Grade Cetuximab Biosimilar) (R&D Systems, Minneapolis, MN, USA) were used as the tumor cell capture agents immobilized on the surface of the microchannels.

Techniques: Control

Journal: Journal of Biological Chemistry

Article Title: Engagement of the Small GTPase Rab31 Protein and Its Effector, Early Endosome Antigen 1, Is Important for Trafficking of the Ligand-bound Epidermal Growth Factor Receptor from the Early to the Late Endosome

doi: 10.1074/jbc.m114.548321

Figure Lengend Snippet: FIGURE 1. Loss of Rab31 hinders endocytic trafficking of the ligand-bound EGFR. A and B, A431 cells were transfected with scrambled (Scr) or Rab31 siRNA and analyzed for EGF trafficking after 48 h. A, top panel, the extent of Rab31 knockdown was assessed by RT-PCR. The endogenous levels of Rab31 protein were low, and Western blot analysis was not useful. Center panel, cells were pulsed with 0.5 g/ml EGF-FITC (green), followed by a 10-min chase before fixation and immunofluorescence analysis with the colabeling of early the endosome marker EEA1 (red). The individual and merged fluorescence signals of the areas enclosedinwhitesquaresareshownenlarged2ontheright.Scalebar20m.Bottompanel,thenumberofEGF-FITCpunctapositiveforEEA1wasquantified and presented graphically as a percentage of total EGF-FITC puncta counted. 27 cells in three independent experiments were analyzed, and data are shown as mean S.E. B, cells were pulsed with 0.5 g/ml EGF-TxR (red) followed by a 30-min chase before fixation and immunofluorescence analysis with the colabeling of the late endosome marker CD63 (green). Top panel, arrowheads show EGF-TxR puncta colocalizing with CD63. Scale bar 20 m. Bottom panel, the number of EGF-TxR puncta also positive for CD63 was quantified and presented graphically as a percentage of total EGF-TxR puncta counted. 27 cells in three independent experiments were analyzed, and data are shown as mean S.E. **, p 0.01; Student’s t test. C, HeLa cells were transfected with scrambled or Rab31siRNA,andanalysiswasperformedafter48h.Leftpanel,knockdownofRab31inHeLacellswasquantifiedbyWesternblotanalysis.Rightpanel,cellswere pulsed with 0.5 g/ml EGF-FITC followed by a 30-min chase before fixation and immunofluorescence analysis with the colabeling of the late endosome marker CD63. The number of EGF-FITC puncta also positive for CD63 was quantified and presented graphically as a percentage of total EGF-FITC puncta counted. 35 cells in three independent experiments were analyzed, and data are shown as mean S.E. **, p 0.01; Student’s t test. D, HeLa cells overexpressing Rab31 (Rab31 OE) and those that were transfected with scrambled or Rab31 siRNA were pulsed with 0.5 g/ml EGF. At the various time points indicated, lysates were harvested and probed by Western blot analysis for total EGFR and -tubulin. Shown is a representative set of data from six independent experiments. Levels of EGFR were normalized against -tubulin and plotted graphically as a percentage of EGFR at 0 min after pulse. *, p 0.05 between scrambled and Rab31 siRNA or scrambled and Rab31 OE at 30 min.

Article Snippet: The following commercial primary antibodies were used: EGFR (Merck Millipore, Singapore), EEA1 (BD Biosciences), GAPex5 (Abcam, Cambridge, UK), RIN3 (Abnova, Taipei, Taiwan), the trans-Golgi marker TGN46 (AbD Serotec, Kidlington, UK), Rab11 (Abcam), and -tubulin (Sigma).

Techniques: Transfection, Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Marker, Fluorescence

Journal: Journal of Biological Chemistry

Article Title: Engagement of the Small GTPase Rab31 Protein and Its Effector, Early Endosome Antigen 1, Is Important for Trafficking of the Ligand-bound Epidermal Growth Factor Receptor from the Early to the Late Endosome

doi: 10.1074/jbc.m114.548321

Figure Lengend Snippet: FIGURE 4. Rab31 associates with EGFR in a high molecular weight complex that includes its effector EEA1 but not Rab5. A, EGFR was coimmunoprecipi- tated (IP) with Rab31 or Rab5 antibody (ab), respectively, using 1 mg of lysates from cells transfected with vector alone (Vector ctrl) and Rab31 (Rab31 OE), respectively, with or without GTPS loading. B, 1 mg of A431 cell lysate (left panel) or HeLa cell lysate (right panel) with and without GTPS was incubated with 20 g of GST or GST-Rab31 and glutathione beads, and the ability of the GST fusion proteins to pull down EGFR was analyzed by Western blot analysis. The GST fusion proteins were visualized with Ponceau S stain. C, 1 mg of A431 cell lysate with and without GTPS was incubated with 20 g of GST-Rab31 or GST-Rab31S19N (S19N) and glutathione beads, and the ability of the GST fusion proteins to pull down EGFR was analyzed by Western blot analysis. The GST proteins were visualized with Ponceau S stain. D, A431 cells stably transfected with EGFP-tagged Rab31 (EGFP-Rab31) were pulsed with 0.5 g/ml EGF-TxR, fixed after 30 min, and analyzed for colocalization between EGFP-Rab31 (green) and EGF-TxR (red). Bottom panel, the boxed areas in the upper panel, enlarged 2. Arrows indicate structures positive for both EGFP-Rab31 and EGF-TxR. Scale bar 20 m. E, the percentage of EGF-TxR-positive puncta that are also positive for EGFP-Rab31 was quantified from cells fixed after a 0-, 10-, and 30-min chase and graphically represented as a percentage of total EGF-TxR puncta counted. 34 cells in three independent experiments were analyzed, and data are shown as mean S.E. **, p 0.01; Student’s t test. F, 2 mg of A431 Rab31 OE lysates with and without GTPS loading was resolved by glycerol gradient sedimentation. Fractions were collected, and TCA was precipitated and analyzed by Western blot analysis for Rab31, EGFR, Rab5, and EEA1 (an effector for both Rab5 and Rab31). The membrane blot for Rab5 was also overexposed. The position in the gradient that contains the molecular size markers BSA (67 kDa), catalase (240 kDa), and thyroglobulin (660 kDa) is indicated.

Article Snippet: The following commercial primary antibodies were used: EGFR (Merck Millipore, Singapore), EEA1 (BD Biosciences), GAPex5 (Abcam, Cambridge, UK), RIN3 (Abnova, Taipei, Taiwan), the trans-Golgi marker TGN46 (AbD Serotec, Kidlington, UK), Rab11 (Abcam), and -tubulin (Sigma).

Techniques: High Molecular Weight, Transfection, Plasmid Preparation, Incubation, Western Blot, Staining, Stable Transfection, Sedimentation, Membrane

Journal: Journal of Biological Chemistry

Article Title: Engagement of the Small GTPase Rab31 Protein and Its Effector, Early Endosome Antigen 1, Is Important for Trafficking of the Ligand-bound Epidermal Growth Factor Receptor from the Early to the Late Endosome

doi: 10.1074/jbc.m114.548321

Figure Lengend Snippet: FIGURE6.EEA1mediatestheRab31-EGFRinteractioninthetraffickingcomplex.A,toppanel,A431EGFP-Rab31OEcells(green)werepulsedwith0.5g/ml EGF-TxR (red), fixed at various time points, and colabeled using EEA1 antibodies (blue). Arrows indicate some structures positive for EGFP-Rab31, EGF-TxR, and EEA1. Scale bar 20 m. Bottom panel, the percentage of EEA1 puncta that are positive for Rab31 was quantified from cells fixed after a 0-, 10-, and 30-min chase and presented graphically as a percentage of total EGF-TxR puncta counted. 33 cells in three independent experiments were analyzed, and data are shown as mean S.E. **, p 0.01; Student’s t test. B, A431 cells were transfected with scrambled (Scr) or EEA1 siRNA. 1 mg of lysates with and without GTPS was incubated with 20 g of GST-Rab31 and glutathione beads. The ability of GST-Rab31 to pull down EGFR was analyzed by Western blot analysis. Ponceau S staining of the GST proteins used is shown. C, cells were pulsed with 0.5 g/ml EGF before harvesting after a 30-min chase. 1 mg of lysates with and without GTPS was incubated with 20 g of GST-Rab31 and glutathione beads. The ability of GST-Rab31 to pull down EGFR and EEA1 was analyzed by Western blot analysis. Ponceau S staining of the GST proteins used is shown. D, A431 EGFP-Rab31 OE cells (green) were transfected with scrambled or EEA1 siRNA. Cells were pulsed 48 h later with 0.5 g/ml EGF-TxR (red), fixed after 30 min, and colabeled for EEA1 (blue). Top panels, Knockdown of EEA1 was assessed by immunofluo- rescence (left) and Western blot analysis (right). Arrowheads also indicate points of colocalization between EGFP-Rab31, EGF-TxR, and EEA1 puncta. Scale bar 20 m. Bottom panels, orthogonal projection of the three-dimensional stacked confocal images shown in the top left panel. In cells transfected with scrambled siRNA, there is colocalization between EGFP-Rab31 (green) and EGF-TxR (red) (arrows). In cells with EEA1 knockdown, EGFP-Rab31 and EGF-TxR appeared delocalized (arrowheads). E, the percentage of EGF-TxR-positive puncta that are also positive for EGFP-Rab31 was quantified from scrambled and EEA1 siRNA-transfected cells fixed after a 0-, 10-, and 30-min chase and graphically represented as a percentage of total EGF-TxR puncta counted. 29 cells in three independent experiments were analyzed, and data are shown as mean S.E. **, p 0.01; Student’s t test.

Article Snippet: The following commercial primary antibodies were used: EGFR (Merck Millipore, Singapore), EEA1 (BD Biosciences), GAPex5 (Abcam, Cambridge, UK), RIN3 (Abnova, Taipei, Taiwan), the trans-Golgi marker TGN46 (AbD Serotec, Kidlington, UK), Rab11 (Abcam), and -tubulin (Sigma).

Techniques: Transfection, Incubation, Western Blot, Staining, Knockdown

Journal: Journal of Biological Chemistry

Article Title: Engagement of the Small GTPase Rab31 Protein and Its Effector, Early Endosome Antigen 1, Is Important for Trafficking of the Ligand-bound Epidermal Growth Factor Receptor from the Early to the Late Endosome

doi: 10.1074/jbc.m114.548321

Figure Lengend Snippet: FIGURE 7. EEA1 is important for the Rab31-mediated enhancement of ligand-bound EGFR endocytic trafficking. A, A431 EGFP-Rab31 OE cells were transfected with scrambled (Scr) or EEA1 siRNA and analyzed after 48 h. Top panel, the levels of EEA1 and EGFR were analyzed by Western blot analysis. Bottom panel, cells were pulsed with 0.5 g/ml EGF-TxR (red) and fixed at 30 min for comparison of the puncta sizes between cells overexpressing EGFP-Rab31 (green, asterisks) and non-overexpressing cells in the same populations.Scale bar 20 m. B, left panels, scrambled and EEA1 siRNA-treated cells were immunostained for CD63 (pseudocolored green) along with EGF-TxR (red) and EGFP-Rab31 (pseudocolored white). Left lower panels, individual and merged fluorescence signals of the boxed areas, magnified 2. Box A represents cells with Rab31 overexpression, whereas box B represents cells with no overexpression. Arrows indicate some structures positive for both EGF-TxR and CD63. Scale bar 20 m. Right panel, the percentage of EGF-TxR puncta that are positive for CD63 were quantified and graphically represented as a percentage of total EGF-TxR puncta counted. 36 cells in three independent experiments were analyzed, and data are shown as mean S.E. **, p 0.01; Student’s t test.

Article Snippet: The following commercial primary antibodies were used: EGFR (Merck Millipore, Singapore), EEA1 (BD Biosciences), GAPex5 (Abcam, Cambridge, UK), RIN3 (Abnova, Taipei, Taiwan), the trans-Golgi marker TGN46 (AbD Serotec, Kidlington, UK), Rab11 (Abcam), and -tubulin (Sigma).

Techniques: Transfection, Western Blot, Comparison, Fluorescence, Over Expression

Journal: Journal of Biological Chemistry

Article Title: Engagement of the Small GTPase Rab31 Protein and Its Effector, Early Endosome Antigen 1, Is Important for Trafficking of the Ligand-bound Epidermal Growth Factor Receptor from the Early to the Late Endosome

doi: 10.1074/jbc.m114.548321

Figure Lengend Snippet: FIGURE 8. The guanine nucleotide exchange factor GAPex5 is important for Rab31-EGFR interaction. A, A431 Rab31 OE cells were transfected with scrambled (Scr), GAPex5, or RIN3 siRNA and assayed after 48 h. Cell lysates were analyzed by Western blot analysis for GAPex5 knockdown and RT-PCR for RIN3 knockdown. Cells were fixed and immunostained for localization of EGFP-Rab31 (green) and TGN46 (red), a marker for the trans-Golgi network. Scale bar 20 m. Colocalization between EGFP-Rab31 and TGN46 was quantified using Zen 2010 software for calculation of the overlap coefficient. 27 cells in three independent experiments were analyzed, and data are shown as mean S.E. *, p 0.05; Student’s t test. B, A431 cells were transfected with GAPex5 siRNA and harvested 48 h later. Left panel, the extent of GAPex5 knockdown and the levels of the EGFR were assessed by Western blot analysis. Right panel, a GST-Rab31 affinity pulldown assay was performed with the harvested lysates. Cells were pulsed with 0.5 g/ml EGF before harvesting after a 30-min chase. 1 mg of lysates with and without GTPS was incubated with 20 g of GST-Rab31 and glutathione beads. The ability of GST-Rab31 to pull down EGFR and EEA1 was analyzed by Western blot analysis. Ponceau S staining of the GST proteins used is shown. C, 1 mg of A431 cell lysate harvested 48 h after transfection with relevant siRNA was incubated with 20 g of GST or GST-Rab31 and glutathione beads in the presence of GTPS, and the ability of the fusion proteins to pull down the EGFR was analyzed by Western blot analysis. GST proteins were visualized with Ponceau S stain.

Article Snippet: The following commercial primary antibodies were used: EGFR (Merck Millipore, Singapore), EEA1 (BD Biosciences), GAPex5 (Abcam, Cambridge, UK), RIN3 (Abnova, Taipei, Taiwan), the trans-Golgi marker TGN46 (AbD Serotec, Kidlington, UK), Rab11 (Abcam), and -tubulin (Sigma).

Techniques: Transfection, Western Blot, Knockdown, Reverse Transcription Polymerase Chain Reaction, Marker, Software, Incubation, Staining