Journal: Molecular oncology

Article Title: A patient tumor transplant model of squamous cell cancer identifies PI3K inhibitors as candidate therapeutics in defined molecular bins

doi: 10.1016/j.molonc.2013.03.004

Figure Lengend Snippet: Assessment of engrafted tumors, A. Schema representing the model. For implantation we used our standard technique (Jimeno et al., 2009; Rubio‐Viqueira et al., 2006), which maintains a narrow control on size and number of pieces of the first (F1) implantation. The number of pieces implanted (6, 8, and 10 tumor pieces in 9%, 22%, and 69% of cases) in F1 did not influence the success of engraftment. B. Orthotopic tumors with locoregional nodes (in the base of tongue [BOT] model the neck is dissected for clarity). Nodes from mice with orthotopic tumors grew >5 mm, compared with <1 mm in normal nodes. Immunohistochemistry (x40; bar 50 μM) with human EGFR and cytokeratin 5, mouse CD45, and dual FISH labeling of mouse cot (green) and humand cot (red) on neck nodes from mice that never had a human tumor (above), and a floor‐of‐mouth (FOM) implantation (below). C. Paired microphotographs (x20; bar 100 μM) of F0 and F2 hematoxilyn‐eosin and EGFR staining of three cases with well (above), moderate (middle) and poor (lower) degree of squamous differentiation. The tumors propagated on mice maintained these features and the EGFR staining across the spectrum. D. All cases showed EGFR positivity by immunohistochemistry (IHC), whose intensity, percentage and H‐score ranged from 1 to 3 (average±SD, 2.5 ± 0.6), 50%–100% (89% ± 18%), and 100 to 300 (226 ± 71). The plot of the correlation between the F0 and F2 H‐score for EGFR staining shows a high degree of correlation (R2 = 0.91, P < 0.0001). Although there were divergences in the H‐score in 9 of 25 paired samples, all were <20% and all cases were classified in the same category. E. Gene expression variations between F0 vs F2 vs F4 generations were compared for the CUHN014 and CUHN022 cases using the expression value of genes that significantly mapped to the human genome in at least one case and/or generation. The greatest variation for both cases was between the F0 patient sample and the F2, with stabilization further. Expression nearly completely stabilized once established on mice with R‐squared values for F2 vs F4 of 0.938 and 0.959 for CUHN014 and CUHN022.

Article Snippet: The primary antibodies for IHC were: EGFR, pAKT (Ser473), pS6, PTEN (Cell Signaling Technology).

Techniques: Immunohistochemistry, Labeling, Staining, Expressing

Journal: Nature Communications

Article Title: LC3B is an RNA-binding protein to trigger rapid mRNA degradation during autophagy

doi: 10.1038/s41467-022-29139-1

Figure Lengend Snippet: a – d CLIP-seq of endogenous LC3B on HEK293T cells treated with either DMSO or Rapa + CQ. Data obtained from two biological replicates of LC3B CLIP-seq were analyzed. The mRNAs with FPKM ≥ 1 in the DMSO-treated cells were used for the calculations. a The number of LC3B CLIP peaks in mRNAs. b Metagene profiles of LC3B peaks. Each region of the 5′UTR, CDS, or 3′UTR was binned into 50 segments. c Consensus motif for LC3B binding. The consensus RNA sequences from LC3B peaks located in the 3′UTR in the cells treated with either DMSO (left) or Rapa + CQ (right) were predicted by MEME (upper) or HOMER (lower). The E value (upper) estimates the expected number of motifs with a similarly sized set of random sequences using log likelihood ratio. The p values (lower) were calculated using cumulative binomial distributions. d Profiles of the distance between LC3B peaks located at the consensus AAUAAA motif and polyadenylation signal (PAS). p values were calculated using the two-tailed Kolmogorov–Smirnov test. e EMSA analysis using in vitro-synthesized Cy5-labeled triple repeats (×3) of either AAUAAA or AAAAAA and either purified recombinant LC3B-WT or BSA. The relative positions of the free probes and RNA-protein complexes are indicated using arrows. Representative data obtained from two independently performed biological replicates ( n = 2) are shown. f Fluorescence polarization assay showing preferential interaction between purified recombinant LC3B and AAUAAA motif. Dissociation constants (Kd) are indicated at the right side of graph; n = 6; Data are presented as mean values ± SD; nd not determined. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against the following proteins were purchased [listed in the format “protein name (catalog number, supplier)”]: LC3B (NB100-2220, Novus; #3868, Cell Signaling Technology; or AM20212PU-N, ORIGENE), CNOT1 (14276-1-AP, Proteintech), CNOT7 (ab195587, Abcam), PRMT1 (07-404, Sigma-Aldrich), FLAG (A8592, Sigma-Aldrich; or F3165, Sigma-Aldrich), p62 (Cell Signaling Technology; 18420-1-AP), G3BP1 (13057-2-AP, Proteintech), GFP (sc-9996, Santa Cruz Biotechnology), U1 snRNP70 (sc-390899, Santa Cruz Biotechnology), β-actin (A5441, Sigma-Aldrich), and GAPDH (LF-PA0212, Ab Frontier).

Techniques: Binding Assay, Two Tailed Test, In Vitro, Synthesized, Labeling, Purification, Recombinant, Fluorescence

Journal: Nature Communications

Article Title: LC3B is an RNA-binding protein to trigger rapid mRNA degradation during autophagy

doi: 10.1038/s41467-022-29139-1

Figure Lengend Snippet: a – c Profiles of mRNA abundance. HEK293T cells either undepleted or depleted of endogenous LC3B were treated with either DMSO or Rapa. Total cell RNAs were purified and subjected to mRNA sequencing. The mRNAs with FPKM ≥ 1 in the cells treated with Control siRNA and DMSO were used for the calculations. a Cumulative distribution function (CDF) plots for relative changes in mRNA abundance after Rapa treatment. The mRNAs were categorized into two groups: total mRNAs either harboring (LC3B CLIP group) or lacking (non-targets group) the LC3B CLIP peak. CDF plots for the relative changes in the abundance of mRNAs upon Rapa treatment in the undepleted cells ( b ) or upon LC3B downregulation in Rapa-treated cells ( c ). The mRNAs belonging to the LC3B CLIP group were categorized into three groups: 5′UTR, CDS, and 3′UTR depending on the position of the LC3B peak. d – g Profiles of mRNA half-life. HEK293T cells either undepleted or depleted of LC3B were treated with either DMSO or Rapa + CQ. The cells were harvested at three time points (0, 6, and 12 h) and total cell RNAs were subjected to mRNA sequencing experiments as described in the “Methods” section. CDF plots for the relative change in the half-life of mRNAs harboring (LC3B CLIP group) or lacking (non-targets group) LC3B peak upon Rapa + CQ treatment in the undepleted cells ( d ) or upon LC3B downregulation in the cells treated with Rapa + CQ ( e ). CDF plots for the relative change in the half-life of mRNAs belonging to the 5′UTR, CDS, or 3′UTR group upon Rapa + CQ treatment in the undepleted cells ( f ) or upon LC3B downregulation in the cells treated with Rapa + CQ ( g ). p values were calculated using the two-tailed Mann–Whitney U test. All NGS data were obtained from two independently performed biological replicates ( n = 2). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against the following proteins were purchased [listed in the format “protein name (catalog number, supplier)”]: LC3B (NB100-2220, Novus; #3868, Cell Signaling Technology; or AM20212PU-N, ORIGENE), CNOT1 (14276-1-AP, Proteintech), CNOT7 (ab195587, Abcam), PRMT1 (07-404, Sigma-Aldrich), FLAG (A8592, Sigma-Aldrich; or F3165, Sigma-Aldrich), p62 (Cell Signaling Technology; 18420-1-AP), G3BP1 (13057-2-AP, Proteintech), GFP (sc-9996, Santa Cruz Biotechnology), U1 snRNP70 (sc-390899, Santa Cruz Biotechnology), β-actin (A5441, Sigma-Aldrich), and GAPDH (LF-PA0212, Ab Frontier).

Techniques: Purification, Sequencing, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: LC3B is an RNA-binding protein to trigger rapid mRNA degradation during autophagy

doi: 10.1038/s41467-022-29139-1

Figure Lengend Snippet: a Read densities of the LC3B CLIP-seq (LC3B CLIP1 and LC3B CLIP2) and mRNA sequencing (input mRNA1 and input mRNA2) for PRMT1 mRNA in two biological replicates. The map for PRMT1 is shown at the bottom. b Schematic representation of RLuc reporter mRNAs harboring the full-length PRMT1 3′UTR of either WT or U/A substitution. The relative positions of the LC3-binding site (AAUAAA) and its variant (AAAAAA) are indicated by arrows. c In vivo CLIP of endogenous LC3B. HEK293T cells expressing the RLuc reporter mRNA and FLuc mRNA (which served as a negative control) were either treated or not treated with Rapa + CQ. The cells were subjected to in vivo CLIP using α-LC3B antibody or nonspecific rabbit IgG (rIgG). The amounts of coimmunoprecipitated reporter mRNAs were normalized to those of the FLuc mRNAs. Then, the normalized levels obtained in IPs using rIgG in the untreated cells were arbitrarily set to 1.0. n = 3; Data are presented as mean values ± SD; p values were analyzed using two-tailed and equal-variance Student’s t test; * p < 0.05; ** p < 0.01 (The exact p values are provided in Source Data file). d Effect of LC3B downregulation on the abundance of the RLuc-P3′ reporter mRNAs. HEK293T cells either undepleted or depleted of LC3B were transiently transfected with plasmids expressing RLuc and FLuc reporter mRNAs. The cells were either treated or not treated with Rapa + CQ for 12 h before cell harvest. The amounts of the RLuc mRNAs were normalized to those of FLuc mRNAs. Then, the normalized levels in the untreated cells were arbitrarily set to 100%; n = 3; Data are presented as mean values ± SD; p values were analyzed using two-tailed and equal-variance Student’s t test; ** p < 0.01 (The exact p values are provided in Source Data file). e Half-life measurement of the RLuc reporter mRNA, RLuc-P3′-WT. As performed in d except that the cells were harvested at the indicated time points. The y axis represents the level of mRNA remaining (percentage) on the logarithmic scale (log2); n = 4; Data are presented as mean values ± SD. f , g Half-life of endogenous LMD substrates after treatment with Rapa + CQ or LC3B downregulation. Two endogenous LMD substrates, PRMT1 mRNA ( f ) and MARS1 mRNA ( g ) were analyzed. n = 3; Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against the following proteins were purchased [listed in the format “protein name (catalog number, supplier)”]: LC3B (NB100-2220, Novus; #3868, Cell Signaling Technology; or AM20212PU-N, ORIGENE), CNOT1 (14276-1-AP, Proteintech), CNOT7 (ab195587, Abcam), PRMT1 (07-404, Sigma-Aldrich), FLAG (A8592, Sigma-Aldrich; or F3165, Sigma-Aldrich), p62 (Cell Signaling Technology; 18420-1-AP), G3BP1 (13057-2-AP, Proteintech), GFP (sc-9996, Santa Cruz Biotechnology), U1 snRNP70 (sc-390899, Santa Cruz Biotechnology), β-actin (A5441, Sigma-Aldrich), and GAPDH (LF-PA0212, Ab Frontier).

Techniques: Sequencing, Binding Assay, Variant Assay, In Vivo, Expressing, Negative Control, Two Tailed Test, Transfection

Journal: Nature Communications

Article Title: LC3B is an RNA-binding protein to trigger rapid mRNA degradation during autophagy

doi: 10.1038/s41467-022-29139-1

Figure Lengend Snippet: a Common features found in LMD substrates. Two common features, (i) a long-range looping and (ii) the secondary or tertiary structures upstream of the AAUAAA motif, are indicated by semi-transparent rectangles. b MFE values (left) and GC content (right) across the nucleotide positions upstream of the AAUAAA motif. The 3′UTR sequences of mRNAs belonging to the 3′UTR_AAUAAA or non-targets group were analyzed. p values were calculated using the two-tailed Kolmogorov–Smirnov test. c Predicted secondary structure of the 3′UTR of PRMT1-WT, M3, and M4. d In vivo CLIPs of endogenous LC3B. As performed in Fig. , except that HEK293T cells were expressed with one of RLuc-P3′ reporter mRNAs (WT, M3, or M4 mRNA). n = 3; Data are presented as mean values ± SD; p values were analyzed using two-tailed and equal-variance Student’s t test; # not significant; * p < 0.05; ** p < 0.01 (The exact p values are provided in Source Data file). e Relative amounts of the RLuc-P3′ reporter mRNAs upon treatment with Rapa + CQ. n = 3; Data are presented as mean values ± SD; p values were analyzed using two-tailed and equal-variance Student’s t test; * p < 0.05; ** p < 0.01 (The exact p values are provided in Source Data file). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against the following proteins were purchased [listed in the format “protein name (catalog number, supplier)”]: LC3B (NB100-2220, Novus; #3868, Cell Signaling Technology; or AM20212PU-N, ORIGENE), CNOT1 (14276-1-AP, Proteintech), CNOT7 (ab195587, Abcam), PRMT1 (07-404, Sigma-Aldrich), FLAG (A8592, Sigma-Aldrich; or F3165, Sigma-Aldrich), p62 (Cell Signaling Technology; 18420-1-AP), G3BP1 (13057-2-AP, Proteintech), GFP (sc-9996, Santa Cruz Biotechnology), U1 snRNP70 (sc-390899, Santa Cruz Biotechnology), β-actin (A5441, Sigma-Aldrich), and GAPDH (LF-PA0212, Ab Frontier).

Techniques: Two Tailed Test, In Vivo

Journal: Nature Communications

Article Title: LC3B is an RNA-binding protein to trigger rapid mRNA degradation during autophagy

doi: 10.1038/s41467-022-29139-1

Figure Lengend Snippet: a Schematic diagram of FLAG-LC3B variants used in this study. b – e Complementation experiments using LC3B variants. HEK293T cells were transiently transfected with either the control siRNA or LC3B siRNA that anneals to 3′UTR of endogenous LC3B mRNA. Two days later, the cells were retransfected with an RLuc-P3′-WT reporter plasmid, a FLuc reference plasmid, and a plasmid expressing either FLAG-LC3B-WT or its variant. The cells were either treated or not treated with Rapa + CQ for 12 h before cell harvest. b , d Western blotting showing specific downregulation of endogenous LC3B and proper expression of FLAG-LC3B or its variant at a level comparable to that of endogenous LC3B. c , e Effect of LC3B-WT or its variant on LMD. The amounts of RLuc mRNAs were normalized to those of FLuc mRNAs. Then, the normalized levels in the untreated cells were arbitrarily set to 100%. n = 3; # not significant; * p < 0.05; ** p < 0.01. f , g Measurement of LMD efficiency in the nucleus and cytoplasm. f Western blotting showing specific separation between the nuclear (N) and cytoplasmic (C) fractions. g Efficiency of LMD of RLuc-P3′-WT reporter mRNAs. n = 3; ** p < 0.01. h Effect of treatment with the indicated chemical inhibitor on LMD of RLuc-P3′-WT reporter mRNAs. n = 3; ** p < 0.01. In c , e , g , h , data are presented as mean values ± SD from three biological replicates. p values were analyzed using two-tailed and equal-variance Student’s t test; The exact p values are provided in Source Data file. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against the following proteins were purchased [listed in the format “protein name (catalog number, supplier)”]: LC3B (NB100-2220, Novus; #3868, Cell Signaling Technology; or AM20212PU-N, ORIGENE), CNOT1 (14276-1-AP, Proteintech), CNOT7 (ab195587, Abcam), PRMT1 (07-404, Sigma-Aldrich), FLAG (A8592, Sigma-Aldrich; or F3165, Sigma-Aldrich), p62 (Cell Signaling Technology; 18420-1-AP), G3BP1 (13057-2-AP, Proteintech), GFP (sc-9996, Santa Cruz Biotechnology), U1 snRNP70 (sc-390899, Santa Cruz Biotechnology), β-actin (A5441, Sigma-Aldrich), and GAPDH (LF-PA0212, Ab Frontier).

Techniques: Transfection, Plasmid Preparation, Expressing, Variant Assay, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: LC3B is an RNA-binding protein to trigger rapid mRNA degradation during autophagy

doi: 10.1038/s41467-022-29139-1

Figure Lengend Snippet: a IPs of endogenous LC3B. The extracts of HEK293T cells either treated or not treated with Rapa + CQ were subjected to IPs using α-LC3B antibody or nonspecific rIgG. The intensities of each western blot image were quantitated. The intensities of coimmunoprecipitated proteins were normalized to those of immunoprecipitated LC3B. The relative levels obtained in untreated cells were arbitrarily set to 1.0. n = 3. b , c IPs of endogenous LC3B upon downregulation of both ATG5 and ATG7. As performed in a , except that the cells were either undepleted or depleted of ATG5 and ATG7. b Western blotting proving specific downregulation of endogenous ATG5 and ATG7. c Western blotting of cellular proteins before or after IPs of LC3B. d The proximity ligation assay (PLA) between endogenous LC3B and either CNOT1 or CNOT7. PLA experiments using the indicated antibodies were performed on HeLa cells treated with either DMSO or Rapa + CQ. The PLA images are shown in Supplementary Fig. . The number of PLA spots per cell was quantified and is presented in this panel. Box-whiskers show maximum, third quartile to first quartile, median and minimum; n = 767 cells examined over three independent experiments. e , f Effect of CNOT1 or CNOT7 downregulation on LMD. e Western blotting showing specific downregulation of endogenous CNOT1 and CNOT7. f LMD efficiency of RLuc-P3′-WT reporter mRNA, endogenous PRMT1 mRNA, and MARS1 mRNA. n = 3; Data are presented as mean values ± SD; p values were analyzed using two-tailed and equal-variance Student’s t test; * p < 0.05; ** p < 0.01 (The exact p values are provided in Source Data file). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against the following proteins were purchased [listed in the format “protein name (catalog number, supplier)”]: LC3B (NB100-2220, Novus; #3868, Cell Signaling Technology; or AM20212PU-N, ORIGENE), CNOT1 (14276-1-AP, Proteintech), CNOT7 (ab195587, Abcam), PRMT1 (07-404, Sigma-Aldrich), FLAG (A8592, Sigma-Aldrich; or F3165, Sigma-Aldrich), p62 (Cell Signaling Technology; 18420-1-AP), G3BP1 (13057-2-AP, Proteintech), GFP (sc-9996, Santa Cruz Biotechnology), U1 snRNP70 (sc-390899, Santa Cruz Biotechnology), β-actin (A5441, Sigma-Aldrich), and GAPDH (LF-PA0212, Ab Frontier).

Techniques: Western Blot, Immunoprecipitation, Proximity Ligation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: LC3B is an RNA-binding protein to trigger rapid mRNA degradation during autophagy

doi: 10.1038/s41467-022-29139-1

Figure Lengend Snippet: a Intracellular distributions of PLA spots involving endogenous LC3B and CNOT1. Intracellular distributions of PLA spots showing the specific interaction between endogenous LC3B and CNOT1 were determined using PLA experiments. In addition, the intracellular distributions of FLAG-LC3B and endogenous p62 (both of which were used for markers for autophagosome), FLAG-DCP1A (a marker for processing bodies), or endogenous G3BP1 (a marker for stress granules) were determined by immunostaining with α-FLAG antibody, α-p62 antibody, α-G3BP1 antibody, and α-G3BP1 antibody, respectively. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). An enlarged view of the white boxed area is provided in the lower right corner of each image. n = 3; Scale bar = 10 μm. b IPs of ATG5, ATG12, and ATG16L1. HEK293T cells were transiently transfected with a plasmid expressing FLAG, FLAG-ATG5, FLAG-ATG12, or FLAG16L1. Two days later, the cells were treated with Rapa + CQ for 12 h before cell harvest. Total cell extracts were subjected to IPs with the α-FLAG antibody; n = 2; Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against the following proteins were purchased [listed in the format “protein name (catalog number, supplier)”]: LC3B (NB100-2220, Novus; #3868, Cell Signaling Technology; or AM20212PU-N, ORIGENE), CNOT1 (14276-1-AP, Proteintech), CNOT7 (ab195587, Abcam), PRMT1 (07-404, Sigma-Aldrich), FLAG (A8592, Sigma-Aldrich; or F3165, Sigma-Aldrich), p62 (Cell Signaling Technology; 18420-1-AP), G3BP1 (13057-2-AP, Proteintech), GFP (sc-9996, Santa Cruz Biotechnology), U1 snRNP70 (sc-390899, Santa Cruz Biotechnology), β-actin (A5441, Sigma-Aldrich), and GAPDH (LF-PA0212, Ab Frontier).

Techniques: Marker, Immunostaining, Staining, Transfection, Plasmid Preparation, Expressing

Journal: Nature Communications

Article Title: LC3B is an RNA-binding protein to trigger rapid mRNA degradation during autophagy

doi: 10.1038/s41467-022-29139-1

Figure Lengend Snippet: a Schematic diagram of siRNA-resistant (R) FLAG-PRMT1 R reporters, WT, M3, or M4 mRNA. These reporter mRNAs are the same as RLuc-P3′-WT, M3, and M4 mRNA described in Fig. , except that the FLAG-PRMT1 R reporter mRNAs encode the full-length PRMT1 protein instead of RLuc. b – d Complementation experiment using the FLAG-PRMT1 R reporter mRNAs. HeLa cells stably expressing GFP-LC3B were transiently transfected with either PRMT1 siRNA or nonspecific Control siRNA. Two days later, the cells were retransfected with a plasmid expressing either FLAG or one of the FLAG-PRMT1 R reporter mRNAs. The cells were treated with Rapa + CQ for 12 h before immunostaining with the α-GFP antibody. n = 3. b Relative level of PRMT1 mRNAs. n = 3. Data are presented as mean values ± SD; # not significant; ** p < 0.01 (The exact p values are provided in Source Data file). c Immunostaining of GFP-LC3B. Scale bar = 10 μm. d Quantitation of GFP-LC3B puncta per cell. Box-whiskers show maximum, third quartile to first quartile, median and minimum. A one-way ANOVA test was conducted to calculate the p values; n = 865 cells examined over three independent experiments; # not significant; ** p < 0.01. e Proposed model illustrating the role of LMD in autophagy. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against the following proteins were purchased [listed in the format “protein name (catalog number, supplier)”]: LC3B (NB100-2220, Novus; #3868, Cell Signaling Technology; or AM20212PU-N, ORIGENE), CNOT1 (14276-1-AP, Proteintech), CNOT7 (ab195587, Abcam), PRMT1 (07-404, Sigma-Aldrich), FLAG (A8592, Sigma-Aldrich; or F3165, Sigma-Aldrich), p62 (Cell Signaling Technology; 18420-1-AP), G3BP1 (13057-2-AP, Proteintech), GFP (sc-9996, Santa Cruz Biotechnology), U1 snRNP70 (sc-390899, Santa Cruz Biotechnology), β-actin (A5441, Sigma-Aldrich), and GAPDH (LF-PA0212, Ab Frontier).

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Immunostaining, Quantitation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: BML-265 and Tyrphostin AG1478 Disperse the Golgi Apparatus and Abolish Protein Transport in Human Cells

doi: 10.3389/fcell.2019.00232

Figure Lengend Snippet: BML-265 and Tyrphostin AG1478 inhibit the transport of secretory proteins. HeLa cells transiently expressing Str-KDEL_TNF-SBP-EGFP (A) , SBP-EGFP-GPI (B) or SBP-EGFP-EGFR (C) were pre-treated with the indicated molecules at 10 μM for 1 h. Trafficking of the reporters was then induced by incubation with biotin for 1 h. The presence of the GFP-tagged reporters (green, upper panel) at the plasma membrane was then detected using an anti-GFP antibody on non-permeabilized cells (red, upper panel). The Golgi apparatus was visualized using immunostaining against GM130 (bottom panel). Scale bar: 10 μm.

Article Snippet: Antibodies to detect phosphorylated EGFR and total EGFR were purchased from Cell Signaling Technology (PhosphoPlus EGFR (Tyr1068) Antibody Duet, catalog number 11862S, dilution 1:1000).

Techniques: Expressing, Incubation, Immunostaining

Journal: Journal of Cell Science

Article Title: Stress-specific p38 MAPK activation is sufficient to drive EGFR endocytosis but not its nuclear translocation

doi: 10.1242/jcs.202358

Figure Lengend Snippet: Immunofluorescence of permeabilized HeLa and SCC47 cells does not reveal increased nuclear EGFR following X-ray treatment. (A) HeLa cells were serum starved, treated with a single dose of X-rays (4 Gy) and then incubated for 30 min before fixation, permeabilization and staining with anti-EGFR extracellular domain (green) and cytoplasmic domain (red) antibodies, and DAPI (blue). Although a small amount of EGFR staining overlaps with DAPI-stained nuclei this is present in both control and X-ray-treated cells. (B) SCC47 cells were serum starved and left untreated, incubated with EGF for 30 min or were X-ray treated (4 Gy) followed by a 30 min incubation before fixation, permeabilization and staining with anti-EGFR cytoplasmic domain antibody (green) and Hoechst 33342 (blue). EGFR is distributed in puncta throughout the cells after EGF stimulation but remains predominantly associated with the plasma membrane following X-ray treatment. Scale bars: 10 μm.

Article Snippet: Anti-EGFR antibodies used for immunofluorescence were against the extracellular domain antibody purified from the mouse 108 hybridoma (ATCC; 1:200), and rabbit anti-cytoplasmic domain antibodies (4267 Cell Signaling; 1:50).

Techniques: Immunofluorescence, Incubation, Staining, Control, Clinical Proteomics, Membrane

Journal: Journal of Cell Science

Article Title: Stress-specific p38 MAPK activation is sufficient to drive EGFR endocytosis but not its nuclear translocation

doi: 10.1242/jcs.202358

Figure Lengend Snippet: Apparent increase in nuclear staining of EGFR on sections of X-ray-treated cells. (A) HeLa cells were serum starved, and treated with or without a single dose of X-rays (4 Gy) followed by a 30 min incubation. Cells were then fixed, cryo-preserved and embedded, and semi-thin cryosections were labeled with anti-EGFR extracellular domain or cytoplasmic domain antibodies (green) and DAPI (blue). Clear nuclear staining is evident with the extracellular domain antibody that is increased following X-ray treatment and absent after labeling with anti-cytoplasmic domain or no primary antibody. (B) HeLa cells treated with control or EGFR-targeting siRNA were western blotted with anti-EGFR antibody to assess efficiency of knockdown or were treated with X-rays (4 Gy) and stained with anti-extracellular domain antibody as in A. Although plasma membrane staining was greatly reduced following EGFR depletion, nuclear staining with anti-extracellular domain antibody was unaffected. (C) NIH 3T3 cells lacking EGFR were treated with X-rays and stained as in A. Nuclear but not plasma membrane staining was clearly present on sections stained with extracellular but not cytoplasmic domain antibody. Scale bars: 10 μm.

Article Snippet: Anti-EGFR antibodies used for immunofluorescence were against the extracellular domain antibody purified from the mouse 108 hybridoma (ATCC; 1:200), and rabbit anti-cytoplasmic domain antibodies (4267 Cell Signaling; 1:50).

Techniques: Staining, Incubation, Labeling, Control, Western Blot, Knockdown, Clinical Proteomics, Membrane

Journal: Journal of Biological Chemistry

Article Title: CFBP Is a Novel Tyrosine-phosphorylated Protein That Might Function as a Regulator of CIN85/CD2AP

doi: 10.1074/jbc.m605693200

Figure Lengend Snippet: FIGURE2.IdentificationofCD2APasabindingproteintoCFBP.A,purificationoftheCFBPcomplex.TheFLAG-taggedCFBPwaspurifiedfromHEK293Tcells transfected with the expression plasmid (right lane). As a control, mock-transfected HEK293T cells transfected with the empty vector were used (left lane). Affinity-purified protein eluted with 0.1 M glycine-HCl buffer (pH 3.0) was analyzed by SDS-PAGE. Purified CFBP and co-precipitated bands (80 kDa) indicated by an arrow and thick line were in-gel digested, and peptides were analyzed by LC/MS/MS. B, sequences in CD2AP identified by LC/MS/MS are boxed. C, association between CFBP and both CD2AP and CIN85 in vivo. Immunoprecipitation (IP) experiments were carried out with HEK293 cell lysates using control IgG, anti-CFBP(C-ter) antibody. The lysates were prepared from cells with () or without () EGF stimulation. Immunoblot (IB) analysis was carried out with anti-CD2AP (-CD2AP) (top), -CIN85 (middle), and -CFBP(His) (bottom) antibodies. D, confirmation of this association in a reciprocal experiment. The preparation of cell lysate was performed as described in B. Immunoprecipitation and immunoblot experiments were performed with the indicated antibodies. CBB, Coomassie Brilliant Blue; WCL, whole cell lysate.

Article Snippet: Other antibodies were obtained commercially from various suppliers: anti-FLAG M2 antibody (Sigma), anti-phosphotyrosine antibody (4G10; Upstate Biotechnology, Inc., Lake Placid, NY), anti-GFP antibody (Molecular Probes, Inc., Eugene, OR), antiCD2AP antibody (SC-9137; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Cbl antibody (SC-1651; Santa Cruz Biotechnology), anti-CIN85 antibody (Calbiochem), anti-EGF receptor antibody (SC-03; Santa Cruz Biotechnology), anti- - actin antibody (Sigma), and anti-HA antibody (12CA5; Roche Applied Science).

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Affinity Purification, SDS Page, Purification, Liquid Chromatography with Mass Spectroscopy, In Vivo, Immunoprecipitation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: CFBP Is a Novel Tyrosine-phosphorylated Protein That Might Function as a Regulator of CIN85/CD2AP

doi: 10.1074/jbc.m605693200

Figure Lengend Snippet: FIGURE 4. Tyrosine 204 of CFBP is the major phosphorylation site upon EGF stimulation. A, tyrosine phosphorylation of endogenous CFBP upon EGF stimulation. CFBP was immunoprecipitated (IP) using anti- CFBP(C-ter) antibody with () or without () EGF stimulation. The control experiment was carried out using control IgG. Immunoblot analysis was carried out using -CFBP(His) (lower panel) or -phosphotyrosine (upper panel).Thepositionsofeachmoleculeareindicatedbyarrows.B,screeningofthetyrosinephosphorylationsite of CFBP upon EGF stimulation. Schematic representation of the wild-type and the mutants of CFBP-L used. The positions of all six tyrosine residues in CFBP are indicated. The numbers indicate the amino acid residues. COS7 cells were transfected with each plasmid encoding the FLAG-tagged construct of CFBP, and immunoprecipi- tation (IP) of the expressed molecule was carried out from the lysate of the cells treated with EGF. Immunoblot analysis was carried out using -FLAG and -phosphotyrosine. The results of tyrosine phosphorylation of CFBP derivatives are indicated as () or () at the left side of each schematic diagram. C, Tyr204 is a tyrosine phos- phorylation site of CFBP upon EGF stimulation. The preparation of lysate of COS7 cells transfected with the expression plasmid encoding FLAG-tagged wild-type or the Y204F mutant of CFBP-L was carried out as describedinB.Immunoprecipitationandimmunoblotwereperformedwiththeindicatedantibodies.D,detec- tion of the phosphorylation at Tyr204 of CFBP by phospho-specific antibody. COS7 cells were transfected with the expression plasmid described above, and EGF treatment and immunoprecipitation were performed as described above. Immunoblot analysis was carried out using the anti-FLAG (lower panel) and anti-phospho- Tyr204 (-pY204) (upper panel) antibodies. E, association of CD2AP/CIN85 and phosphorylated CFBP upon EGF stimulation. A time course experiment of the phosphorylation at Tyr204 and association of the molecules was analyzed by immunoprecipitation and immunoblot. COS7 cells transfected with FLAG-tagged CFBP-S were treated with EGF for the indicated times, and immunoprecipitation was performed as described above. Immu- noblot analysis was carried out using the indicated antibodies. F, association of CFBP and CIN85/CD2AP is dependent on the phosphorylation at Tyr204 of CFBP. COS7 cells expressing FLAG-tagged CFBP-S derivatives were treated with EGF for 10 min, and immunoprecipitation was performed as described above. Immunoblot analysis was carried out using the indicated antibodies.

Article Snippet: Other antibodies were obtained commercially from various suppliers: anti-FLAG M2 antibody (Sigma), anti-phosphotyrosine antibody (4G10; Upstate Biotechnology, Inc., Lake Placid, NY), anti-GFP antibody (Molecular Probes, Inc., Eugene, OR), antiCD2AP antibody (SC-9137; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Cbl antibody (SC-1651; Santa Cruz Biotechnology), anti-CIN85 antibody (Calbiochem), anti-EGF receptor antibody (SC-03; Santa Cruz Biotechnology), anti- - actin antibody (Sigma), and anti-HA antibody (12CA5; Roche Applied Science).

Techniques: Phospho-proteomics, Immunoprecipitation, Control, Western Blot, Transfection, Plasmid Preparation, Construct, Expressing, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: CFBP Is a Novel Tyrosine-phosphorylated Protein That Might Function as a Regulator of CIN85/CD2AP

doi: 10.1074/jbc.m605693200

Figure Lengend Snippet: FIGURE 5. Co-localization of CFBP with CD2AP and EGF receptor in cultured cells. Subcellular localization of CFBP was traced by immunofluorescence staining and GFP-fused protein. Exogenous CFBP and endoge- nous CD2AP co-localize in HEK293T cells. Cells were transfected with the expression vector encoding FLAG epitope-tagged CFBP. Cells were processed for co-immunofluorescence staining 48 h post-transfection using -FLAG (a) and -CD2AP (b) antibodies and followed by Alexa-fluor 488-conjugated goat anti-mouse IgG and Alexa-fluor 592-conjugated goat anti-rabbit IgG as the secondary antibodies, respectively. The merged image of a and b is shown in c. CFBP and CD2AP co-localize in actin-enriched structures. COS7 cells were co-trans- fected with the plasmids for expression of GFP-fused CFBP (d) and FLAG-tagged CD2AP (e). Cells were pro- cessed for immunofluorescence staining using anti-FLAG antibody (e). The GFP-CFBP image is shown in d, and the merged image of d and e is shown in f. The mutant CFBP without the proline-rich motif does not co-localize with CD2AP. COS7 cells were transfected with a plasmid for the expression of FLAG-tagged CFBPPro. Cells were processed for immunofluorescence staining using anti-FLAG antibody (g), rhodamine phalloidin for staining actin (h), and Hoechst 33342 for staining the nucleus (i). CFBP co-localizes with EGF receptor. COS7 cells were co-transfected with plasmids for expression of FLAG-tagged CD2AP and EGF receptor. Cells were processed for immunofluorescence staining using anti-FLAG antibody (j) and anti-EGF receptor antibody (k), The merged image of j and k is shown in l. Scale bars, 10 m.

Article Snippet: Other antibodies were obtained commercially from various suppliers: anti-FLAG M2 antibody (Sigma), anti-phosphotyrosine antibody (4G10; Upstate Biotechnology, Inc., Lake Placid, NY), anti-GFP antibody (Molecular Probes, Inc., Eugene, OR), antiCD2AP antibody (SC-9137; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Cbl antibody (SC-1651; Santa Cruz Biotechnology), anti-CIN85 antibody (Calbiochem), anti-EGF receptor antibody (SC-03; Santa Cruz Biotechnology), anti- - actin antibody (Sigma), and anti-HA antibody (12CA5; Roche Applied Science).

Techniques: Cell Culture, Immunofluorescence, Staining, Transfection, Expressing, Plasmid Preparation, FLAG-tag, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: CFBP Is a Novel Tyrosine-phosphorylated Protein That Might Function as a Regulator of CIN85/CD2AP

doi: 10.1074/jbc.m605693200

Figure Lengend Snippet: FIGURE 6. The CFBP and CIN85 complex facilitates the down-regulation of the EGF receptor (EGFR). A, comparison of the remaining EGF receptor by immunofluorescence staining in two independent COS7 cells in the image with (upper) or without (lower) expression of FLAG-CFBP-S after 2 h of EGF treatment. Cells were processed for co-immunofluorescence staining using anti-FLAG (left) and anti-EGF receptor (right) antibodies. Scale bars, 10 m. B, effect of the expression of CFBP in response to EGF stimulation. Comparison of the amount of each protein was monitored by immunoblot (IB) with specific antibodies. Twenty micrograms of protein of each whole cell lysate prepared from COS7 cells expressing each CFBP derivative stimulated with EGF for the indicated times was applied. C, effect of siRNA knockdown of CFBP on the response to EGF stimulation detected by immunoblot. Comparison of the amount of the EGF receptor (top), CFBP (middle), and -actin (bottom) was monitored by immunoblot with each specific antibody. Twenty micrograms of protein of each wholecelllysatepreparedfromvector-infected(leftthreelanes)andsiRNAconstruct-infected(rightthreelanes) HeLa cells stimulated with EGF for the indicated times was applied in each lane. D, CFBP forms a ternary complex with CIN85 and Cbl; the effect of the expression of CFBP on the CIN85-Cbl complex is shown. The complex was monitored by immunoprecipitation (IP) in COS7 cells co-expressing HA-tagged Cbl and the indicated CFBP derivative. Cells with EGF stimulation were lysed, and the immunoprecipitation was performed with anti-HA antibody. Immunoprecipitated Cbl, co-precipitated CIN85, and CFBP are indicated in the middle, upper, and lower panels, respectively.

Article Snippet: Other antibodies were obtained commercially from various suppliers: anti-FLAG M2 antibody (Sigma), anti-phosphotyrosine antibody (4G10; Upstate Biotechnology, Inc., Lake Placid, NY), anti-GFP antibody (Molecular Probes, Inc., Eugene, OR), antiCD2AP antibody (SC-9137; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Cbl antibody (SC-1651; Santa Cruz Biotechnology), anti-CIN85 antibody (Calbiochem), anti-EGF receptor antibody (SC-03; Santa Cruz Biotechnology), anti- - actin antibody (Sigma), and anti-HA antibody (12CA5; Roche Applied Science).

Techniques: Comparison, Immunofluorescence, Staining, Expressing, Western Blot, Knockdown, Infection, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: CFBP Is a Novel Tyrosine-phosphorylated Protein That Might Function as a Regulator of CIN85/CD2AP

doi: 10.1074/jbc.m605693200

Figure Lengend Snippet: FIGURE 7. Ectopic expression of CFBP induces morphological change in A431 cells. A, comparison of the protein expression levels of EGF receptor (EGFR; top), CD2AP (middle), and CFBP (bottom) in various mammalian cultured cells. Each whole cell lysate (WCL) prepared from the indicated cells was subjected to SDS-PAGE and analyzed by immunoblot (IB) with each antibody. HEK293T cells were transfected with the empty vector (first lane from the left) or the expression vectors for overexpressing the recombinant proteins of EGF receptor and CFBP-S (second lane from the left). B, protein expression levels in stably expressing FLAG-CFBP-S A431 cells. Two independent representative clones (clones C8 and E3) stably expressing FLAG-CFBP are shown. Control cells were mock-transfected with the empty vector. For monitoring the amount of proteins, 4 g (for the EGF receptor) or 20 g of protein (for CD2AP and CFBP) of whole cell lysate was used and analyzed by immunoblot using each specific antibody. C, effect of the expression of CFBP in response to EGF stimulation. Comparison of the amount of each protein was monitored by immunoblot with specific antibodies. Twenty micrograms of protein of each whole cell lysate prepared from control (left panel set) and clone E3 (right panel set) cells stimulated with EGF for the indicated times was applied. D, densitometric analysis of data in C. The results represent three independent experiments. Error bars show the S.D. The amounts of EGF receptor at each time point are normalized to the control (lane of each 0 time). E, effect of the expression of CFBP on the CD2AP-Cbl complex. The complex was monitored by immunoprecipitation in A431 cell lines. The control and E3 cell lines with or without EGF stimulation were lysed, and the immunopre- cipitation was performed with anti-CD2AP antibody. Immunoprecipitated CD2AP and co-precipitated Cbl are indicated in the lower and upper panels, respectively. F, expression of CFBP induced morphological alteration of A431 cells. Images show representative colonies, each comprising of 10–20 cells of the A431 cell line. Images were taken at 30 h of the last passage of the cells on the culture dish in normal growth medium. Images of control A431 and the A431 clone C8 cells stably expressing FLAG-CFBP-S are shown in the top and bottom rows of panels, respectively. The left, middle, and right columns show the images of phase contrast, actin, and the nucleus, respectively. Scale bar, 50 m. G, graph showing the average area of each cell in the colony. This value was calculated by measuring the area of a colony in (F) and divided by the number of cells contained within that colony. Data were taken from 30 randomly selected colonies in one experiment, and three independent sets of experiments were carried out. Error bars, S.D.

Article Snippet: Other antibodies were obtained commercially from various suppliers: anti-FLAG M2 antibody (Sigma), anti-phosphotyrosine antibody (4G10; Upstate Biotechnology, Inc., Lake Placid, NY), anti-GFP antibody (Molecular Probes, Inc., Eugene, OR), antiCD2AP antibody (SC-9137; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Cbl antibody (SC-1651; Santa Cruz Biotechnology), anti-CIN85 antibody (Calbiochem), anti-EGF receptor antibody (SC-03; Santa Cruz Biotechnology), anti- - actin antibody (Sigma), and anti-HA antibody (12CA5; Roche Applied Science).

Techniques: Expressing, Comparison, Cell Culture, SDS Page, Western Blot, Transfection, Plasmid Preparation, Recombinant, Stable Transfection, Clone Assay, Control, Immunoprecipitation

Journal: The Journal of Neuroscience

Article Title: Lysosomal Dysfunction in Down Syndrome Is APP-Dependent and Mediated by APP-βCTF (C99)

doi: 10.1523/JNEUROSCI.0578-19.2019

Figure Lengend Snippet: Decreased lysosomal turnover of autophagic protein substrates in DS fibroblasts. A–D, Fibroblasts from 3 different DS patients and age-matched control were labeled for 2 d with [3H] leucine. After extensive washing, cells were plated in complete medium (A, B) or in serum-depleted media (C, D); and where indicated, (B, D) 20 mm NH4Cl/100 μm leupeptin (NL) was added into the incubation medium. The rate of total protein degradation at the indicated times was calculated as the percentage of total radiolabeled protein transformed in soluble amino acids and small peptides at each time. Values are the mean ± SEM (n = 10 replicates). E, Western blotting of autophagy-related proteins in 2N and DS fibroblasts treated with vehicle or AZD (0.5 μm for 2 h) and with or without leupeptin and pepstatin A (LP, 10 μm for 24 h), with actin as an internal control, and (F) quantification of LC3-II protein levels normalized with actin in 2N and DS fibroblasts (n = 6 replicates). G, Representative confocal microscopic images and (H) the size quantification of lysosome identified by dextran-AlexaFluor-546 uptake (4 h pulse and 20 h chase) in 2N and DS fibroblasts with or without AZD treatment shown as scatter plot (n ≥ 52 lysosomes). Red lines indicate mean values. I, Quantifications of size and number of endosomes and lysosomes in 2N and DS fibroblasts (n ≥ 30 cells). J, Representative EM images of HRP uptake in 2N and DS fibroblasts and (K) the quantification of HRP-positive lysosomes at 1 and 24 h uptake time point (n ≥ 9 field). Arrows indicate HRP-positive vesicles. + sign indicates mean in Bar and Whiskers plots. n.s., not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005. Scale bar, 5 μm.

Article Snippet: Antiserum to RAB7 (ab50533) was from Abcam, EEA1 (610456) from BD Biosciences, and LC3 (NB100-2220) from Novus; antibodies to total p-70 (9202) and pThr389-p70 (9205) were from Cell Signaling Technology, Lamp2 (H4B4) from Developmental Studies Hybridoma Bank, cathepsin B (CTSB, GT15047) from Neuromics ( Wolfe et al., 2013 ), cathepsin D (CTSD) from Scripps Institute (90236), antibodies against APP, APP-CTFs (C1/6.1, 6E10, N25), and CTSD (CD1.1) have been described previously ( Mathews et al., 2002 ), actin (A2066) and GAPDH (G9545) from Sigma-Aldrich/Millipore.

Techniques: Labeling, Incubation, Transformation Assay, Western Blot

Journal: The Journal of Neuroscience

Article Title: Lysosomal Dysfunction in Down Syndrome Is APP-Dependent and Mediated by APP-βCTF (C99)

doi: 10.1523/JNEUROSCI.0578-19.2019

Figure Lengend Snippet: Lysosomal acidification deficiency in DS fibroblasts. A, Representative confocal microscopic images of 2N and DS fibroblasts transfected with mRFP-eGFP-LC3 for 48 h and dextran-AlexaFluor-647 uptake as lysosome marker and (B) less acidic ALs as a percentage of total ALs (n ≥ 31 cells). Arrows indicate a less acidic AL. + sign indicates mean in Bar and Whiskers plot. ***p ≤ 0.005. Scale bar, 5 μm.

Article Snippet: Antiserum to RAB7 (ab50533) was from Abcam, EEA1 (610456) from BD Biosciences, and LC3 (NB100-2220) from Novus; antibodies to total p-70 (9202) and pThr389-p70 (9205) were from Cell Signaling Technology, Lamp2 (H4B4) from Developmental Studies Hybridoma Bank, cathepsin B (CTSB, GT15047) from Neuromics ( Wolfe et al., 2013 ), cathepsin D (CTSD) from Scripps Institute (90236), antibodies against APP, APP-CTFs (C1/6.1, 6E10, N25), and CTSD (CD1.1) have been described previously ( Mathews et al., 2002 ), actin (A2066) and GAPDH (G9545) from Sigma-Aldrich/Millipore.

Techniques: Transfection, Marker

Journal: Cancer Research

Article Title: Heparanase Augments Epidermal Growth Factor Receptor Phosphorylation: Correlation with Head and Neck Tumor Progression

doi: 10.1158/0008-5472.can-08-2910

Figure Lengend Snippet: Figure 1. Heparanase modulates EGFR phosphorylation levels—gene transfection. LNCaP prostate carcinoma (A), Daoy medulloblastoma (B), U87 glioma (C), and A431 epidermoid carcinoma (D) cells (top) were transfected with heparanase cDNA (Hepa) or control, empty vector (Vo). Enhanced heparanase expression was revealed by activity assay (top; x, control Vo cells; n, heparanase-transfected cells) and immunoblotting, applying anti-heparanase antibody (second panels, Hepa). EGFR phosphorylation was examined by subjecting cell lysates to IP with anti-PY antibody, followed by immunoblotting with anti-EGFR antibody (A, third panel), or by using the reciprocal experiment (IP for EGFR, blot with anti-PY; fourth panel). Elevation of EGFR phosphorylation after heparanase transfection was also revealed by immunoblotting with anti phospho-EGFR (Tyr1173) antibody (A, bottom), and this antibody was further used to examine EGFR phosphorylation in Daoy (B), U87 (C), and A431 (D) cells (third panels, p-EGFR). Fourth panels, equal EGFR loading (B–D; EGFR). EGFR phosphorylation index was calculated by densitometry analysis of phosphorylated EGFR (Tyr1173) levels divided by the total EGFR values. Data are presented as fold increase of EGFR phosphorylation compared with control, Vo cells, set arbitrarily to a value of 1 (B–D, bottom).

Article Snippet: Polyclonal antibodies to phospho-Src (Tyr416), phospho-Akt (Ser473), and phospho-EGFR (Tyr845, Tyr1068, Tyr1148) were purchased from Cell Signaling.

Techniques: Phospho-proteomics, Transfection, Control, Plasmid Preparation, Expressing, Activity Assay, Western Blot

Journal: Cancer Research

Article Title: Heparanase Augments Epidermal Growth Factor Receptor Phosphorylation: Correlation with Head and Neck Tumor Progression

doi: 10.1158/0008-5472.can-08-2910

Figure Lengend Snippet: Figure 2. Heparanase modulates EGFR phosphorylation levels—exogenous addition and gene silencing. A, immunostaining. Control, Vo, and heparanase-transfected A431 cells (Hepa, top), or 5-Am sections of tumor xenograft produced by these cells (second panels) were subjected to immunofluorescent (top) or immunohistochemistry staining with anti phospho-EGFR antibody (Tyr1173). B, heparanase addition. U87 glioma cells were left untreated as control (0) or incubated with recombinant 65 kDa heparanase protein (WT; 1 Ag/mL) for the time indicated (min; left). Cell lysates were subjected to immunoblotting with anti–phospho-EGFR (Tyr1173; top), anti-EGFR (second panel), anti–phospho-Akt (p-Akt; third panel), and anti-Akt (fourth panel) antibodies. Right, elevation of EGFR phosphorylation (Tyr1173) after exogenous addition of DM, inactive heparanase (1 Ag/mL). C, heparanase gene silencing. LNCaP prostate carcinoma cells were left untreated as control (Con) or transfected with anti-GFP or anti-heparanase siRNA oligonucleotides. Total RNA was extracted 2 d after transfection and heparanase expression was examined by reverse transcription-PCR analysis (top). GAPDH transcript was used as an internal control for RNA loading (second panel). Cell lysates were prepared from corresponding cultures and subjected to immunoblotting with anti–phospho-EGFR (Tyr1173; third panel) and anti-EGFR (fourth panel) antibodies. Bottom, densitometry analysis of EGFR phosphorylation levels after anti-heparanase siRNA treatment. Control cells were arbitrarily set to a value of 1.

Article Snippet: Polyclonal antibodies to phospho-Src (Tyr416), phospho-Akt (Ser473), and phospho-EGFR (Tyr845, Tyr1068, Tyr1148) were purchased from Cell Signaling.

Techniques: Phospho-proteomics, Immunostaining, Control, Transfection, Produced, Immunohistochemistry, Staining, Incubation, Recombinant, Western Blot, Expressing, Reverse Transcription

Journal: Cancer Research

Article Title: Heparanase Augments Epidermal Growth Factor Receptor Phosphorylation: Correlation with Head and Neck Tumor Progression

doi: 10.1158/0008-5472.can-08-2910

Figure Lengend Snippet: Figure 3. EGFR activation by heparanase is mediated via Src. A, U87 cells were transfected with control empty vector (Vo), or plasmids encoding wild-type heparanase, heparanase lacking the heparin-binding domain (Gln270-Lys280; D10) that results in accumulation of heparanase in the culture medium (18), or lacking the heparin binding domain (Lys158-Asn171; D15), resulting in a nonsecreted protein (18). Cell lysates were subjected to immunoblotting with anti–phospho-EGFR (Tyr1173; top), anti-EGFR (second panel), anti–phospho-Src (Tyr416; third panel), and anti-Src (bottom) antibodies. Note that enhanced EGFR and Src phosphorylation requires heparanase secretion. B, control (Vo) and heparanase-transfected (Hepa) LNCaP cells were left untreated (0) or incubated with the Src inhibitor PP2 (5 Amol/L). Cell lysates were subjected to immunoblotting with anti–phospho-EGFR (Tyr1173; top), anti-EGFR (second panel), anti–phospho-Src (Tyr416; third panel), and anti-Src (fourth panel) antibodies. EGFR and Src phosphorylation levels were similarly evaluated in control untreated parental LNCaP cells (Con) and after addition of the wild-type (WT, middle) or DM (right) heparanase proteins, without (0) or with 5 Amol/L PP2 (5). C, gene silencing. LNCaP cells were transfected with anti-GFP or anti-heparanase siRNA oligonucleotides, and cell lysates were subjected to immunoblotting with anti–phospho-Src (p-Src; top) and anti-Src (bottom) antibodies. Note decreased Src phosphorylation after heparanase down-regulation. Src gene silencing. Heparanase-transfected LNCaP cells were transfected anti-GFP or anti-Src siRNAs, and cell lysates were subjected to immunoblotting applying anti–phospho-Src (p-Src, top), anti-Src (second panel), anti–phospho-EGFR (1173, third panel), anti–phospho-EGFR (845, fourth panel), and anti-EGFR (bottom) antibodies (middle). Parental LNCaP cells were similarly transfected with anti-GFP or anti-Src siRNA oligonucleotides and incubated (30 min, 37jC) without () or with (+) purified recombinant heparanase protein. Cell lysates were subjected to immunoblotting applying the above mentioned antibodies (right). D, heparanase-transfected LNCaP cells were untreated (Hepa) or incubated with the indicated concentration of Src inhibitor PP2 (Amol/L) for 30 min. Mock-transfected cells were used as control (Vo). Cell lysates were then subjected to immunoblotting with anti–phospho-EGFR (Tyr1173; top), anti–phospho-EGFR (Tyr845; second panel), anti-EGFR (third panel), anti–phospho-Src (Tyr416; fourth panel), and anti-Src (bottom) antibodies.

Article Snippet: Polyclonal antibodies to phospho-Src (Tyr416), phospho-Akt (Ser473), and phospho-EGFR (Tyr845, Tyr1068, Tyr1148) were purchased from Cell Signaling.

Techniques: Activation Assay, Transfection, Control, Plasmid Preparation, Binding Assay, Western Blot, Phospho-proteomics, Incubation, Purification, Recombinant, Concentration Assay

Journal: Cancer Research

Article Title: Heparanase Augments Epidermal Growth Factor Receptor Phosphorylation: Correlation with Head and Neck Tumor Progression

doi: 10.1158/0008-5472.can-08-2910

Figure Lengend Snippet: Figure 4. Heparanase modulates cell proliferation. A, heparanase-transfected LNCaP cells were plated at 5 104 cells per dish without (Hepa) or with EGFR inhibitor (Hepa+1478; 5 Amol/L), and cell number was determined 3, 5, and 7 d after plating by a Coulter counter and hemacytometer. Mock-transfected cells (Vo) incubated without or with the EGFR inhibitor (Vo+1478; 5 Amol/L) were used as control. B, BrdUrd incorporation. Direct measurement of DNA synthesis is shown by BrdUrd incorporation. Subconfluent cultures of control (Vo) and heparanase-transfected (Hepa) LNCaP cells were grown in serum-free medium for 20 h followed by incubation with BrdUrd (1:1,000) for 2 h. Cells were then fixed and immunostained with anti-BrdUrd monoclonal antibodies. Positively stained, red-brown nuclei were counted versus blue, hematoxilin counter-stained nuclei. At least 1,000 cells were counted for each cell type, and the percentage of positively stained cells is noted in each panel. C, colony formation in soft agar. Control (Vo) and heparanase-transfected LNCaP cells (5 103 cells per dish) were mixed with soft agar and cultured for 3 wk in the absence (Hepa) or presence of Src (Hepa+PP2; 5 Amol/L) or EGFR (Hepa+1478; 10 Amol/L) inhibitors. Shown are photomicrographs of colonies at low (10; top) and high (100; bottom) magnification. D, gene silencing. LNCaP (left), MDA-MB-231 (middle), and U87 (right) cells were transfected with anti–GFP (si-GFP; top) or anti-heparanase (si-Hepa; bottom) siRNA oligonucleotides and BrdUrd incorporation was evaluated as above, except that cells were not serum-starved. Note 2.5- to 4-fold decrease in cell proliferation after heparanase gene silencing.

Article Snippet: Polyclonal antibodies to phospho-Src (Tyr416), phospho-Akt (Ser473), and phospho-EGFR (Tyr845, Tyr1068, Tyr1148) were purchased from Cell Signaling.

Techniques: Transfection, Incubation, Control, DNA Synthesis, Bioprocessing, Staining, Cell Culture

Journal: Cancer Research

Article Title: Heparanase Augments Epidermal Growth Factor Receptor Phosphorylation: Correlation with Head and Neck Tumor Progression

doi: 10.1158/0008-5472.can-08-2910

Figure Lengend Snippet: Figure 5. Tumor xenograft and biopsy specimens. U87 cells transfected with control empty vector (Vo) or heparanase plasmid (Hepa) were inoculated s.c. (5 106/ 0.1 mL), and tumor volume was measured twice a week starting on day 21 (A). At the end of the experiment on day 35, tumors were resected, weighted (A, inset) and fixed in formalin. Paraffin-embedded 5-Am sections were stained with anti–phospho-EGFR (Tyr1173; B, bottom) and anti–phospho-Src (Tyr416; B, top) antibodies. C, immunohistochemical staining of phospho-EGFR in human head and neck tumor specimens. Formalin-fixed, paraffin-embedded 5-Am sections of head and neck tumors were subjected to immunostaining of phosphorylated EGFR, applying anti–phospho-EGFR (Tyr1173) antibody, as described under Materials and Methods. Shown are representative photomicrographs of phospho-EGFR–negative (top), and positively stained specimens scored as weak (+1; second panel) and strong (+2; third panel) intensity.

Article Snippet: Polyclonal antibodies to phospho-Src (Tyr416), phospho-Akt (Ser473), and phospho-EGFR (Tyr845, Tyr1068, Tyr1148) were purchased from Cell Signaling.

Techniques: Transfection, Control, Plasmid Preparation, Staining, Immunohistochemical staining, Formalin-fixed Paraffin-Embedded, Immunostaining

Journal: eLife

Article Title: Ezrin defines TSC complex activation at endosomal compartments through EGFR–AKT signaling

doi: 10.7554/eLife.98523

Figure Lengend Snippet:

Article Snippet: For western blot analysis, the following antibodies were used: mouse anti-NBR1 (1:1000, Abnova MO1), rabbit anti-LAMP1 (1:500, Sigma L1418), mouse anti-Ezrin (1:1000, Novex 357300), mouse anti-SQSTM1/P62 (1:1000, Abcam ab56416), rabbit anti-Cathepsin D (1:1000, Cell Signaling 2284), rabbit anti-LC3 (1:1000, Novus NB100-2220), mouse anti-GAPDH (1:1000, Santa Cruz SC-32233), rabbit anti-HER2/ErbB2 (1:1000, Cell Signaling 2165), rabbit anti-HER3/ErbB3 (1:1000, Cell Signaling 12708), rabbit anti-phospho-EGF receptor (Tyr845) (1:1000, Cell Signaling 6963), rabbit anti-EGF receptor (1:1000, Cell Signaling 4267), rabbit anti-MAPKAPK-2 (1:1000, Cell Signaling 3042), rabbit anti-phospho-MAPKAPK-2 (Thr222) (1:1000, Cell Signaling 3316), rabbit anti-p38 MAPK (1:1000, Cell Signaling 8690), rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (1:1000, Cell Signaling 4511), rabbit anti-ZO1 (1:1000, Abcam ab216880), mouse anti-EEA1 (1:1000, BD 610457), rabbit anti-Tuberin/TSC2 (1:1000, Cell Signaling 4308), rabbit anti-phospho-Tuberin/TSC2 (Ser939) (1:1000, Cell Signaling 3615), rabbit anti-phospho-Tuberin/TSC2 (Thr1462) (1:1000, Cell Signaling 3617), rabbit anti-p70 S6 Kinase (1:1000, Cell Signaling 9202), mouse anti-phospho-p70 S6 Kinase (Thr389) (1:1000, Cell Signaling 9206), rabbit anti-Akt (1:1000, Cell Signaling 9272), rabbit anti-phospho-Akt (Ser473) (1:1000, Cell Signaling 4060), rabbit anti-4E-BP1 (1:1000, Cell Signaling 9644), rabbit anti-phospho-4E-BP1 (Ser65) (1:1000, Cell Signaling 9456), rabbit anti-phospho-4E-BP1 (Thr37/46) (1:1000, Cell Signaling 2855), rabbit anti-Hamartin/TSC1 (1:1000, Cell Signaling 6935), mouse anti-EGFR (1:500, Santa Cruz sc-120), mouse anti-p-EGFR (1:500, Santa Cruz sc-57542).

Techniques: CRISPR, Transfection, Construct, Sequencing, Recombinant, Purification, Western Blot, Software, Mass Spectrometry, Immunofluorescence, Functional Assay, Fluorescence, Imaging, Staining, Plasmid Preparation