Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: Identification of variants binding HER3. ( A ) Binding activity of TK-hu A3 and TK-hu A4 antibody variants to human HER3, as measured by ELISA. Six combinations of TK-hu A3 heavy (H) and light (L) chains and nine combinations of TK-hu A4 variants were transiently expressed in HEK293 cells. Supernatants were harvested 72 h post-transfection and assessed for HER3 binding. Bars represent mean absorbance ± SD of triplicate measurements. H/L ratio: DNA heavy chain to DNA light chain ratio. ( B ) Expression and integrity of recombinant mAbs H1L1 (lane 1), H2L1 (lane 2) from TK-hu A3, and H3L1 (lane 3) from TK-hu A4 were analyzed by SDS-PAGE under reducing conditions (1 mM DTT) followed by Coomassie blue staining. Molecular weight markers corresponding to the heavy chain (~50 kDa) and light chain (~25 kDa) are indicated.

Article Snippet: Membranes were blocked with 5% nonfat dry milk and 0.1% Tween 20 (Merck KGaA) in Tris-buffered saline (TBS), pH 7.4, and incubated overnight at 4 °C with antibodies against phospho HER3-Tyr1289 (code #2842, Cell Signaling Technology, Danvers, MA, USA), phospo AKT-Ser473 (code #4060, Cell Signaling Technology), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (code #4377, Cell Signaling Technology), HER3 (code #4754, Cell Signaling Technology, ), AKT (code #4691, Cell Signaling Technology), p44/42 MAPK (Erk1/2) (code #4695, Cell Signaling Technology).

Techniques: Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Recombinant, SDS Page, Staining, Molecular Weight

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: Antibodies species cross-reactivity. Binding of TK-murine A3, TK-hu A3, and TK-hu A4 purified antibody variants to recombinant mouse, rat, human, and rhesus HER3 was determined in a dose–response experiment by ELISA. Data represent mean absorbance ± SD from triplicate wells.

Article Snippet: Membranes were blocked with 5% nonfat dry milk and 0.1% Tween 20 (Merck KGaA) in Tris-buffered saline (TBS), pH 7.4, and incubated overnight at 4 °C with antibodies against phospho HER3-Tyr1289 (code #2842, Cell Signaling Technology, Danvers, MA, USA), phospo AKT-Ser473 (code #4060, Cell Signaling Technology), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (code #4377, Cell Signaling Technology), HER3 (code #4754, Cell Signaling Technology, ), AKT (code #4691, Cell Signaling Technology), p44/42 MAPK (Erk1/2) (code #4695, Cell Signaling Technology).

Techniques: Binding Assay, Purification, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: Binding activity of TK-hu A3 and TK-hu A4 antibody variants. ( A ) TK-murine A3 and TK-murine A4 antibodies, as well as TK-hu A3 and TK-hu A4 purified antibody variants, were analyzed for their binding to members of the ErbB tyrosine kinase receptor family: EGFR, ErbB2, HER3, and ErbB4. Antibodies were used at a concentration of 10 µg/mL, and binding was analyzed by ELISA. Absorbance values are plotted on the y-axis with respect to the antibody tested on the different recombinant proteins. ( B ) Serial dilutions of TK-murine A3, TK-hu A3, and TK-hu A4 purified antibody variants were analyzed by ELISA to assess their binding to human HER3 in a dose-dependent manner. Absorbance values are plotted on the y-axis with respect to the antibody tested at different dilutions.

Article Snippet: Membranes were blocked with 5% nonfat dry milk and 0.1% Tween 20 (Merck KGaA) in Tris-buffered saline (TBS), pH 7.4, and incubated overnight at 4 °C with antibodies against phospho HER3-Tyr1289 (code #2842, Cell Signaling Technology, Danvers, MA, USA), phospo AKT-Ser473 (code #4060, Cell Signaling Technology), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (code #4377, Cell Signaling Technology), HER3 (code #4754, Cell Signaling Technology, ), AKT (code #4691, Cell Signaling Technology), p44/42 MAPK (Erk1/2) (code #4695, Cell Signaling Technology).

Techniques: Binding Assay, Activity Assay, Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: Epitope mapping of murine and humanized anti-HER3 antibodies. ( A ) Peptide array binding assays for TK-hu A3-H1L1, TK-hu A3-H2L1, and the murine antibody TK-A3. Reactivity was measured by ELISA using overlapping 15-mer peptides covering the HER3 extracellular domain (ECD). Each colored bar represents the optical density (OD) at 405 nm for an individual peptide. Strong binding was observed for peptides #54 and #55 in TK-hu A3-H1L1 and TK-A3, indicating conserved epitope recognition. In contrast, TK-hu A3-H2L1 showed loss of binding to these peptides and gained reactivity for peptide #66, suggesting a shift in epitope specificity. C+ = positive control; C− = negative control. ( B ) Structural mapping of the identified epitopes onto the HER3 ECD (PDB entry: 1M6B). Left: location of peptide #54 and #55 (orange) shared by TK-A3 and TK-hu A3-H1L1. Right: location of peptide #66 (purple), recognized exclusively by TK-hu A3-H2L1. Protein domains are indicated with Roman numerals. Only HER3 domain II is rendered as surface. ( C ) Amino acid sequences of the relevant peptides with conserved regions highlighted in bold. Peptides #54 and #55 share the core motif HCFGPNPNQCC, while peptide #66 contains a distinct sequence (QPLVYNKLTFQLEPN), reflecting altered antigen recognition in TK-hu A3-H2L1.

Article Snippet: Membranes were blocked with 5% nonfat dry milk and 0.1% Tween 20 (Merck KGaA) in Tris-buffered saline (TBS), pH 7.4, and incubated overnight at 4 °C with antibodies against phospho HER3-Tyr1289 (code #2842, Cell Signaling Technology, Danvers, MA, USA), phospo AKT-Ser473 (code #4060, Cell Signaling Technology), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (code #4377, Cell Signaling Technology), HER3 (code #4754, Cell Signaling Technology, ), AKT (code #4691, Cell Signaling Technology), p44/42 MAPK (Erk1/2) (code #4695, Cell Signaling Technology).

Techniques: Peptide Microarray, Binding Assay, Enzyme-linked Immunosorbent Assay, Positive Control, Negative Control, Sequencing

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: Structure of HER3::TK-hu A3 Fab complex. ( A ) Front view (left) and side view (right) of HER3 (cyan) in complex with TK-hu A3 Fab (light chain, V L , in light green and heavy chain, V H , in coral). Only HER3 domain II is rendered as surface; domains I–IV are indicated with Roman numerals. The domain II dimerization arm (residues 242–257, chain B) is shown in magenta. HER3 domain II surface colored by Fab contacts (atom–atom cutoff 4.0 Å). ( B ) Residues contacting V L only are light green, residues contacting V H only are coral, and residues contacting both chains are yellow. The HER3 cartoon is shown underneath (cyan); the Fab is omitted for clarity. (Contact patches computed with PDBePISA). Atomic details of HER3::TK-hu A3 Fab interface region. ( C , D ) TK-hu A3 Fab binds to the N-terminus region of domain II of HER3 mostly via hydrogen bonds, except for R84, which is involved in a π-cation interaction with residue Y33/F. ( C ): interactions between V L (variable light chain, light green) and HER3 domain II (cyan). ( D ): interactions between V H (variable heavy chain, coral) and HER3 domain II (cyan) (PDB entry: 9I1Q).

Article Snippet: Membranes were blocked with 5% nonfat dry milk and 0.1% Tween 20 (Merck KGaA) in Tris-buffered saline (TBS), pH 7.4, and incubated overnight at 4 °C with antibodies against phospho HER3-Tyr1289 (code #2842, Cell Signaling Technology, Danvers, MA, USA), phospo AKT-Ser473 (code #4060, Cell Signaling Technology), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (code #4377, Cell Signaling Technology), HER3 (code #4754, Cell Signaling Technology, ), AKT (code #4691, Cell Signaling Technology), p44/42 MAPK (Erk1/2) (code #4695, Cell Signaling Technology).

Techniques: Residue

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: TK-hu A3 and TK-hu A4 antibodies variants bind HER3 receptor in its native conformation. MCF7 cells were incubated on ice for 1 h in the presence or absence of TK-murine A3 ( A ), TK-hu A3-H1L1 ( B ), TK-hu A3-H2L1 ( C ), and TK-hu A4-H3L1 ( D ) antibody variants at concentrations of 10 µg/mL and 100 µg/mL. After incubation, cells were washed, and antibody binding was detected using anti-mouse or anti-human IgG antibody conjugated to Alexa Fluor 488 (gray bars). Cells were analyzed using a CytoFLEX flow cytometer platform. Histograms display the percentage of HER3-positive cells. CTRL antibody: The APC anti-human HER3/HER-3 Antibody was used as a positive control (white bars).

Article Snippet: Membranes were blocked with 5% nonfat dry milk and 0.1% Tween 20 (Merck KGaA) in Tris-buffered saline (TBS), pH 7.4, and incubated overnight at 4 °C with antibodies against phospho HER3-Tyr1289 (code #2842, Cell Signaling Technology, Danvers, MA, USA), phospo AKT-Ser473 (code #4060, Cell Signaling Technology), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (code #4377, Cell Signaling Technology), HER3 (code #4754, Cell Signaling Technology, ), AKT (code #4691, Cell Signaling Technology), p44/42 MAPK (Erk1/2) (code #4695, Cell Signaling Technology).

Techniques: Incubation, Binding Assay, Flow Cytometry, Positive Control

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: TK-hu A3 and TK-hu A4 antibodies inhibit NRG-driven HER3 signaling and reduce cancer cell viability. ( A ) Neuregulin competition assay. SK-BR-3 cells were incubated for 1 h at 4 °C with increasing concentrations of TK-hu A3 or TK-hu A4, either in the presence or absence of recombinant neuregulin (NRG). Binding to cell-surface HER3 was assessed by flow cytometry. The plot shows representative binding curves: TK-hu A3 ± NRG (purple and yellow), and TK-hu A4 ± NRG (light green and dark green). The percentage of HER3-positive cells is plotted on the y-axis versus antibody concentration (log10 [µg/mL], x-axis). ( B ) Western blot analysis of HER3 signaling pathway activation in MCF7, FaDu, and BxPC-3 cells. Cells were pretreated for 6 h with increasing concentrations of TK-hu A3 or TK-hu A4 antibodies, then stimulated with NRG. The Control (Ctrl) lane represents untreated, unstimulated cells, while the NRG lane represents NRG-stimulated cells in the absence of antibody. Blots were probed for phosphorylated HER3 (pHER3, Tyr1289), total HER3, phosphorylated Akt (pAkt, Ser473), and phosphorylated p42/44 MAPK (Thr202/Tyr204). β-tubulin served as the loading control. ( C ) Colony-formation assay. BxPC-3 cells were seeded in 6-well plates, treated or not with increasing concentrations of TK-hu A3-H1L1 or TK-hu A4-H3L1 and cultured to allow colony formation; colonies were then fixed, stained, and counted. Only colonies comprising >50 cells were scored as survival colonies. The percentage of inhibition of colony formation (% inh) relative to untreated controls is plotted against the logarithmic antibody concentration (log 10 [µg/mL]), and data are fitted with a four-parametric non-linear regression curve (GraphPad v8.0).

Article Snippet: Membranes were blocked with 5% nonfat dry milk and 0.1% Tween 20 (Merck KGaA) in Tris-buffered saline (TBS), pH 7.4, and incubated overnight at 4 °C with antibodies against phospho HER3-Tyr1289 (code #2842, Cell Signaling Technology, Danvers, MA, USA), phospo AKT-Ser473 (code #4060, Cell Signaling Technology), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (code #4377, Cell Signaling Technology), HER3 (code #4754, Cell Signaling Technology, ), AKT (code #4691, Cell Signaling Technology), p44/42 MAPK (Erk1/2) (code #4695, Cell Signaling Technology).

Techniques: Competitive Binding Assay, Incubation, Recombinant, Binding Assay, Flow Cytometry, Concentration Assay, Western Blot, Activation Assay, Control, Colony Assay, Cell Culture, Staining, Inhibition

Journal: Clinical Cancer Research

Article Title: PI3Kα Inhibitor and Degrader Inavolisib Can Co-opt FGFR2 to Enhance Responses in Patients with PIK3CA -Mutated Solid Tumors and in Preclinical Models

doi: 10.1158/1078-0432.CCR-25-1459

Figure Lengend Snippet: Inavolisib sensitivity depends on high FGFR2 expression. A, Legend related to panels in B–I . B, Cell lines were treated with 0.03 μmol/L of the FGFR2i or 2-μmol/L lapatinib for 1 hour followed by immunoblotting with the antibodies indicated at left. Representative results from experiments ( n = 2). C, Cell lines were treated with inavolisib or the FGFR2i at various concentrations for 1 hour. Cell lysates were immunoprecipitated with an antibody against p85β, followed by immunoblotting with the antibodies indicated at left. Representative results from experiments ( n = 2). IB, immunoblotting. D, Following RAS-GTP pulldown, cell lines were treated with the FGFR2i or lapatinib for different durations and immunoblotted with the antibodies indicated at left. Representative results from experiments ( n = 2). E, Cell lysates from cells treated with inavolisib alone or in combination with the FGFR2i or lapatinib for 4 hours were immunoprecipitated with RAS antibody and blotted with p110α antibody. F, Mechanistic model of the effects of FGFR2 and HER2 inhibition on HER3 and RAS activity. FGFR2-high–expressing cell lines induced PI3K signaling through both HER3 and WT RAS activity (top) compared with HER2-induced PI3K signaling through HER3 but not RAS activity (bottom). G, FGFR2-high–expressing cell lines, MFM223 and SUM52PE, were treated with inavolisib, FGFR2i, or lapatinib for 1 hour. Membrane fractions were analyzed by reciprocal co-IP with one another using HER3 or FGFR2 antibody and Western blotting with FGFR2, HER3, RAS, and p85β antibody. Representative results from experiments ( n = 2). H, SUM52PE, MFM223, and MFE280 cells were treated with inavolisib single-agent or in combination with the FGFR2i or lapatinib for 6 hours. Ubiquitinated proteins were pulled down from the membrane fraction with TUBE1 reagent and blotted with p110α antibody. Representative results from experiments ( n = 2). I, Western blots of the inhibitor response in PI3K signaling (pHER3 and pAKT) in PIK3CA mutant MFM223 and PIK3CA WT SUM52PE; cell lines were treated with 0.5-μmol/L inavolisib or 1-μmol/L alpelisib for different durations. Representative results from experiments ( n = 2). J, Ratio of inavolisib and alpelisib GR 50 values in FGFR2-high ( n = 12) vs. FGFR2-low ( n = 9) expressing cell lines harboring PIK3CA mutations, as assessed in a 5-day viability assay. Data are represented as median (center line) ± IQR (25th to 75th percentile, box) and ± full range (minimum to maximum, whiskers). P value was calculated using Wilcoxon rank-sum test. Representative results from experiments ( n = 2). WB, Western blotting.

Article Snippet: Antibodies to p110α (cat. No. 4249, RRID: AB_2165248), pAKT Ser473 (cat. No. 4060, RRID: AB_2315049), pS6 S235/236 (cat. No. 2211, RRID: AB_331679), HER3 (cat. No. 12708, RRID: AB_2721919), HER2 (cat. No. 2242, RRID: AB_331015), pHER2 Y1221/Y1222 (cat. No. 2243, RRID: AB_490899), pHER3 Y128 (cat. No. 4791, RRID: AB_2099709), pHER3 Y1328 (cat. No. 14525, RRID: AB_2798501), pPLCγ Y783 (cat. No. 14008, RRID: AB_2728690), pERK T202/T204 (cat. No. 9101, RRID: AB_331646), p4EBP T37/46 (cat. No. 9459, RRID: AB_330985), FGFR1 (cat. No. 9740, RRID: AB_11178519), FGFR2 (cat. No. 11835, RRID: AB_2797742), FGFR3 (cat. No. 4574, RRID: AB_2246903), FGFR4 (cat. No. 8562, RRID: AB_10891199), pFGFR Y653/654 (cat. No. 3476, RRID: AB_331369), and pFRS2A Y196 (cat. No. 3864, RRID: AB_2106222) were obtained from Cell Signaling Technology.

Techniques: Expressing, Western Blot, Immunoprecipitation, Inhibition, Activity Assay, Membrane, Co-Immunoprecipitation Assay, Mutagenesis, Viability Assay

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Valine on the Synthesis of α-Casein in MAC-T Cells and the Expression and Phosphorylation of Genes Related to the mTOR Signaling Pathway

doi: 10.3390/ijms26073179

Figure Lengend Snippet: Effects of different valine concentrations on the phosphorylation of downstream proteins in the mTOR signaling pathway. ( A ) Western blotting was used to detect the protein expression levels of phosphorylated mTOR (P-mTOR) and phosphorylated S6K1 (P-S6K1). ( B ) The phosphorylation level of mTOR protein (P-mTOR/mTOR) was quantified and calculated using β-actin and mTOR as internal references. ( C ) The phosphorylation level of S6K1 protein (P-S6K1/S6K1) was quantified and calculated using β-actin and S6K1 as internal references. ( D ) Western blotting was used to detect the protein expression levels of phosphorylated 4EBP1 (P-4EBP1) and phosphorylated RPS6 (P-RPS6). ( E ) The phosphorylation level of 4EBP1 protein (P-4EBP1/4EBP1) was quantified and calculated using β-actin and 4EBP1 as internal references. ( F ) The phosphorylation level of RPS6 protein (P-RPS6/RPS6) was quantified and calculated using β-actin and RPS6 as internal references. ( G ) Western blotting was used to detect the protein expression levels of phosphorylated eEF2 (P-eEF2). ( H ) The phosphorylation level of eEF2 protein (P-eEF2/eEF2) was quantified and calculated using β-actin and eEF2 as internal references. The error bars represent the SD ( n = 3). ANOVA was used to detect the differences between groups, and the LSD method was combined for uncorrected exploratory pairwise comparisons, while the Duncan method was used for conservative multiple comparison corrections. Different lowercase letters indicate significant differences ( p < 0.05), while different uppercase letters indicate significant differences ( p < 0.01).

Article Snippet: , RPS6 , biorbyt , orb585017 , 5% egg white , 1:1000.

Techniques: Phospho-proteomics, Western Blot, Expressing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Valine on the Synthesis of α-Casein in MAC-T Cells and the Expression and Phosphorylation of Genes Related to the mTOR Signaling Pathway

doi: 10.3390/ijms26073179

Figure Lengend Snippet: Effects of valine and rapamycin on the phosphorylation levels of mTOR downstream proteins. ( A ) The phosphorylation levels of mTOR (P-mTOR) in MAC-T cells treated with varying concentrations of rapamycin were assessed using Western blotting. ( B ) The P-mTOR level (P-mTOR/mTOR) in MAC-T cells treated with 100 nM rapamycin was quantified and calculated using β-actin and mTOR as internal references. ( C ) Western blotting was used to detect the protein expression levels of P-mTOR and P-S6K1. ( D ) The P-mTOR level (P-mTOR/mTOR) was quantified and calculated using β-actin and mTOR as internal references. ( E ) The P-S6K1 level (P-S6K1/S6K1) was quantified and calculated using β-actin and S6K1 as internal references. ( F ) Western blotting was used to detect the protein expression levels of P-4EBP1 and P-RPS6. ( G ) The P-4EBP1 level (P-4EBP1/4EBP1) was quantified and calculated using β-actin and 4EBP1 as internal references. ( H ) The P-RPS6 level (P-RPS6/RPS6) was quantified and calculated using β-actin and RPS6 as internal references. ( I ) Western blotting was used to detect the protein expression levels of P-eEF2 and α-casein. ( J ) The P-eEF2 level (P-eEF2/eEF2) was quantified and calculated using β-actin and eEF2 as internal references. ( K ) The α-casein protein expression level was quantified using β-actin as an internal reference. The error bars represent the SD ( n = 3). ANOVA was used to detect the differences between groups, and the LSD method was combined for uncorrected exploratory pairwise comparisons, while the Duncan method was used for conservative multiple comparison corrections. Different lowercase letters indicate significant differences ( p < 0.05), while different uppercase letters indicate significant differences ( p < 0.01).

Article Snippet: , RPS6 , biorbyt , orb585017 , 5% egg white , 1:1000.

Techniques: Phospho-proteomics, Western Blot, Expressing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Valine on the Synthesis of α-Casein in MAC-T Cells and the Expression and Phosphorylation of Genes Related to the mTOR Signaling Pathway

doi: 10.3390/ijms26073179

Figure Lengend Snippet: Primer sequences of internal reference genes and target genes.

Article Snippet: , RPS6 , biorbyt , orb585017 , 5% egg white , 1:1000.

Techniques: Sequencing

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Valine on the Synthesis of α-Casein in MAC-T Cells and the Expression and Phosphorylation of Genes Related to the mTOR Signaling Pathway

doi: 10.3390/ijms26073179

Figure Lengend Snippet: Detailed information of antibodies.

Article Snippet: , RPS6 , biorbyt , orb585017 , 5% egg white , 1:1000.

Techniques: