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: Journal of medicinal chemistry

Article Title: Multiplexed Targeting of Barrett’s Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft In Vivo and Human Specimens Ex Vivo

doi: 10.1021/acs.jmedchem.8b00405

Figure Lengend Snippet: On confocal microscopy, fluorescence from binding of candidate heterodimer with linkers A) E2, B) Hex, C) E3, D) E6, and E) E10 to the surface (arrow) of SKBr3 cells can be seen. F) Quantified results show that the E3 linker provides the highest mean fluorescence intensity. P-values were determined using unpaired t-test. Measurements are an average of 10 randomly chosen cells from 4 images collected independently. G) Western blot shows EGFR and ErbB2 expression in SKBr3 and QhTERT cells.

Article Snippet: Anti-EGFR antibody (#2232S), anti-HER2 antibody (#2165), anti-phospho-EGFR sampler kit (#9922s), anti-phospho-HER2/ErbB2 (Tyr1248) antibody (#2247), anti-AKT (#4691P), anti-ERK1/2 (#4695P), anti-phospho-AKT (pS473; #4060P), anti-phospho-ERK1/2 (#4370P) were used per manufacturer’s instructions (Cell Signaling, Inc).

Techniques: Confocal Microscopy, Fluorescence, Binding Assay, Western Blot, Expressing

Journal: Journal of medicinal chemistry

Article Title: Multiplexed Targeting of Barrett’s Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft In Vivo and Human Specimens Ex Vivo

doi: 10.1021/acs.jmedchem.8b00405

Figure Lengend Snippet: On representative confocal microscopy images of human esophageal specimens ex vivo, QRH*-KSP*-E3-Cy5.5 (red) shows minimal staining to A) squamous (SQ) and B) Barrett’s esophagus (BE), and increased intensity with C) high-grade dysplasia (HGD) and D) esophageal adenocarcinoma (EAC). Similar results were found with AF568-labeled anti-EGFR antibody (yellow) and for AF488-labeled anti-ErbB2 antibody (green). Merged images show co-localization of peptide and antibody binding.

Article Snippet: Anti-EGFR antibody (#2232S), anti-HER2 antibody (#2165), anti-phospho-EGFR sampler kit (#9922s), anti-phospho-HER2/ErbB2 (Tyr1248) antibody (#2247), anti-AKT (#4691P), anti-ERK1/2 (#4695P), anti-phospho-AKT (pS473; #4060P), anti-phospho-ERK1/2 (#4370P) were used per manufacturer’s instructions (Cell Signaling, Inc).

Techniques: Confocal Microscopy, Ex Vivo, Staining, Labeling, Binding Assay

Journal: Journal of medicinal chemistry

Article Title: Multiplexed Targeting of Barrett’s Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft In Vivo and Human Specimens Ex Vivo

doi: 10.1021/acs.jmedchem.8b00405

Figure Lengend Snippet: In siRNA knockdown experiments, QRH*-KSP*-E3-Cy5.5 (red) shows significantly greater binding to the surface (arrows) of A) siCL (control) SkBr3 cells compared with that for B) siEGFR (knockdown) cells. Similar results were found for C) siCL and D) siErbB2 (knockdown) cells. E) Quantified results show significantly greater intensity for siCL versus siEGFR and siCL versus siErbB2, P=3.6×10−4 and P=7.8×10−3, respectively, by unpaired t-test. The mean value was calculated from 5 cells chosen randomly from 3 images collected independently. F) Western blot shows EGFR and ErbB2 expression in control and knockdown cells. G) The apparent dissociation constant (binding affinity) for QRH*-KSP*-E3-Cy5.5 was found to be kd = 23 versus 98 and 54 nM for QRH*-Cy5.5 and KSP*-Cy5.5. H) The apparent association time constant for QRH*-KSP*-E3-Cy5.5 was found to be k = 0.22 min−1 (4.5 min) versus 0.21 min−1 (4.8 min) and 0.35 min−1 (2.9 min) for QRH*-Cy5.5 and KSP*-Cy5.5. Results for each measurement are representative of 3 independent experiments.

Article Snippet: Anti-EGFR antibody (#2232S), anti-HER2 antibody (#2165), anti-phospho-EGFR sampler kit (#9922s), anti-phospho-HER2/ErbB2 (Tyr1248) antibody (#2247), anti-AKT (#4691P), anti-ERK1/2 (#4695P), anti-phospho-AKT (pS473; #4060P), anti-phospho-ERK1/2 (#4370P) were used per manufacturer’s instructions (Cell Signaling, Inc).

Techniques: Binding Assay, Western Blot, Expressing

Journal: Journal of medicinal chemistry

Article Title: Multiplexed Targeting of Barrett’s Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft In Vivo and Human Specimens Ex Vivo

doi: 10.1021/acs.jmedchem.8b00405

Figure Lengend Snippet: We evaluated the effect of the QRH*-KSP*-E3-Cy5.5 on downstream cell signaling after binding to SKBr3 cells. On Western blot, we observed no change in phosphorylation of EGFR (p-EGFR), ErbB2 (p-ErbB2) or of downstream AKT (p-AKT) and ERK (p-ERK) with incubation of heterodimer at 1, 5, and 20 μM. By comparison, the addition of EGF, an endogenous ligand for EGFR, showed increased expression of p-AKT and p-ERK. The addition of 100 nM of lapatinib, a tyrosine kinase inhibitor known to interrupt EGFR/ErbB2 signaling in solid tumors, showed reduced expression of p-EGFR, p-ErbB2 and p-AKT. Cells treated with 1% DMSO and untreated cells showed no suppression of EGFR and ErbB2 mediated signaling. β-tubulin is used as loading control.

Article Snippet: Anti-EGFR antibody (#2232S), anti-HER2 antibody (#2165), anti-phospho-EGFR sampler kit (#9922s), anti-phospho-HER2/ErbB2 (Tyr1248) antibody (#2247), anti-AKT (#4691P), anti-ERK1/2 (#4695P), anti-phospho-AKT (pS473; #4060P), anti-phospho-ERK1/2 (#4370P) were used per manufacturer’s instructions (Cell Signaling, Inc).

Techniques: Binding Assay, Western Blot, Incubation, Expressing

Journal: Journal of medicinal chemistry

Article Title: Multiplexed Targeting of Barrett’s Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft In Vivo and Human Specimens Ex Vivo

doi: 10.1021/acs.jmedchem.8b00405

Figure Lengend Snippet: A) On confocal microscopy, serial sections of HGD in human esophageal specimens are shown following staining with QRH*-KSP*-E3-Cy5.5 (red), anti-EGFR antibody labeled with AF568 (yellow) and anti-ErbB2 antibody labeled with AF488 (green). Fluorescence intensities were quantified from the mean of a set of 3 boxes with dimensions of 20×20 μm2 placed over random crypts. Co-localization of binding can be appreciated on the merged image. B) High-magnification images are shown from dashed boxes. On the merged image, Pearson’s correlation coefficient of ρ = 0.60 and 0.75 was measured for EGFR and ErbB2, respectively. C) From n = 31, 8, 23, and 12 specimens of SQ, BE, HGD, and EAC, respectively, we found significantly greater mean fluorescence intensity from HGD and EAC compared with that for BE and SQ with QRH*-KSP*-E3-Cy5.5, the P-value for difference are calculated by Tukey’s multiple comparisons. A similar result was found for anti-EGFR-AF568 and anti-ErbB2-AF488. D) ROC curve shows 88% sensitivity, 87% specificity and 0.95 AUC with QRH*-KSP*-E3-Cy5.5; 74% sensitivity, 69% specificity, and 0.79 AUC with QRH*-Cy5.5, and 85% sensitivity, 79% specificity, and 0.91 AUC with KSP*-Cy5.5.

Article Snippet: Anti-EGFR antibody (#2232S), anti-HER2 antibody (#2165), anti-phospho-EGFR sampler kit (#9922s), anti-phospho-HER2/ErbB2 (Tyr1248) antibody (#2247), anti-AKT (#4691P), anti-ERK1/2 (#4695P), anti-phospho-AKT (pS473; #4060P), anti-phospho-ERK1/2 (#4370P) were used per manufacturer’s instructions (Cell Signaling, Inc).

Techniques: Confocal Microscopy, Staining, Labeling, Fluorescence, Binding Assay

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: JCI Insight

Article Title: DUOX1 mediates persistent epithelial EGFR activation, mucous cell metaplasia, and airway remodeling during allergic asthma

doi: 10.1172/jci.insight.88811

Figure Lengend Snippet: (A) Western blot of phosphorylation status of EGFR and Src in primary cultures of NECs from 7 healthy and 7 asthmatic subjects. (B) RT-PCR analysis of EGFR ligands EGF, AREG, and TGF-α (TGFA) in NECs from healthy (n = 17) and asthmatic (n = 13) subjects; box, 25th–75th quartiles; whiskers, minimums and maximums. *P < 0.01 by 2-tailed unpaired t test. (C) Inverse correlation between percentage of FEV1 and EGFR phosphorylation status (determined by densitometry of pEGFR/EGFR ratio) as well as relative AREG mRNA expression in NECs from asthmatic subjects. (D) Western blot analysis of EGFR and Src phosphorylation in association with cysteine oxidation in cultured NECs from 7 healthy and 7 asthmatic subjects, determined with phospho-specific antibodies and analysis of avidin-purified proteins after DCP-Bio1 derivatization of oxidized cysteines (-SOH). (E and F) Analysis of EGF- or AREG-induced activation and cysteine oxidation in HBE1 cells transfected with either NS siRNA or DUOX1 siRNA (E) or mTECs from wild-type or Duox1–/– mice (F). Representative blots of at least 2 independent experiments are shown.

Article Snippet: Cell lysates or lung homogenates were prepared using Western solubilization buffer (50 mM HEPES, 250 mM NaCl, 1.5 mM MgCl 2 , 1% Triton X-100, 10% glycerol, 1 mM EGTA, 1 mM PMSF, 2 mM Na 3 VO 4 , 10 μg/ml aprotinin, 10 μg/ml leupeptin; pH 7.4), and samples containing equal amounts of protein (measured using the BCA protein assay kit; Pierce) were separated on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with antibodies against EGFR (C74B9; 1:1,000), pEGFR Tyr845 (D63B4; 1:500), pEGFR Tyr1068 (D7A5; 1:1,000), and Src (L4A1; 1:1,000) (all from Cell Signaling); p-Src Tyr416 (1:1,000; 07-909; Millipore); or DUOX1 (provided by F. Miot, Free University of Brussels, Brussels, Belgium) ( 62 ).

Techniques: Western Blot, Phospho-proteomics, Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture, Avidin-Biotin Assay, Purification, Activation Assay, Transfection

Journal: Cell

Article Title: EGFR ligands differentially stabilize receptor dimers to specify signaling kinetics

doi: 10.1016/j.cell.2017.09.017

Figure Lengend Snippet: (A) Representative time-courses of EGFR phosphorylation at Y1173, Y845, and Y1086 in MCF-7 cells induced by saturating levels of EGF (16 nM), EREG (20 μM) or EPGN (20 μM). Anti-Grb2 is used as loading control. Data for pY1173 were generated by stripping and reprobing pY845 blots, so use the same loading controls.

Article Snippet: With the exception of Grb2 (C-23) from Santa Cruz Biotechnology, all antibodies were from Cell Signaling Technology, and were used at 1:1000 unless otherwise noted: phospho-p44/42 MAPK (Erk1/2) pT202/pY204 (E10), Akt pS473 (D9E), EGFR pY1173 (53A5 at 1:800), EGFR pY845 (2231), EGFR pY1086 (2220 at 1:500), EGFR pY1068 (2234 at 1:500), ErbB2 pY1221/pY1222 (6B12 at 1:700), ErbB3 pY1289 (D1B5 at 1:700), ErbB4 pY1284 (21A9 at 1:700), EGFR (D38B1), ErbB2 (D8F12 at 1:700), ErbB3 (D22C5 at 1:700), and ErbB4 (111B2 at 1:700).

Techniques: Generated, Stripping Membranes