Journal: Pathogens

Article Title: Cellular Receptor Tyrosine Kinase Signaling Plays Important Roles in SARS-CoV-2 Infection

doi: 10.3390/pathogens14040333

Figure Lengend Snippet: Western blot analysis of RTK activation in A549-ACE2 cells after SARS-CoV-2 infection. Cell lysates from mock infection (Mock), A/PR8 (PR8), and SARS-CoV-2 infection at different time points were analyzed by Western blotting to detect active and total forms of EGFR ( A ), TrkA ( B ), and HER2 ( C ). Protein bands were quantified using Sciugo software 2.0.1. The ratio of the phosphorylated form to total form was calculated and compared to the mock (set as 1.00).

Article Snippet: The following antibodies were used for immunoblotting: rabbit anti-human TrkA polyclonal antibody (Cell Signaling Technology, 2505S, Danvers, MA, USA), rabbit anti-human pTrkA monoclonal antibody (mAb) (Cell Signaling Tech, 4168S), rabbit anti-human HER2 mAb (Cell Signaling Tech, 2165S), rabbit anti-human pHER2 mAb (Cell Signaling Tech, 2243L), mouse anti-human EGFR mAb (Cell Signaling Tech, 2239S), rabbit anti-human pEGFR mAb (Cell Signaling Tech, 3777S), rabbit anti-human GAPDH mAb (Cell Signaling Tech, 2118S), anti-rabbit HRP conjugated (Bio Rad, 1706515, Hercules, CA, USA), and anti-mouse IgG HRP conjugated (R&D, HAF007).

Techniques: Western Blot, Activation Assay, Infection, Software

Journal: International Journal of Molecular Sciences

Article Title: Molecular Characterization and Inhibition of a Novel Stress-Induced Mitochondrial Protecting Role for Misfolded TrkAIII in Human SH-SY5Y Neuroblastoma Cells

doi: 10.3390/ijms25105475

Figure Lengend Snippet: ( a ) Indirect IFs demonstrating increased overlapping (yellow/orange) immunoreactivity for TrkA (green) and MitoTracker-labeled mitochondria (red) (upper panels) and Y490 phosphorylated TrkAIII (pTrkAIII, green) and MitoTracker-labeled mitochondria (red) (lower panels) in DTT-treated (5 mM for 6 h) TrkAIII SH-SY5Y cells compared to untreated controls (control). DAPI stained nuclei are blue. (bar = 50 μm). ( b ) Western blots demonstrating TrkAIII cleavage and Y674/5 phosphorylation in mitochondria (50 mg) from DTT-treated (5 mM for 6 h) TrkAIII SH-SY5Y cells compared to untreated TrkAIII SH-SY5Y controls. ( c ) Western blots demonstrating increased PTPase oxidation (arrows) in mitochondria (50 mg) from DTT-treated TrkAIII SH-SY5Y cells (5 mM for 6 h) compared to untreated controls (Con). ( d ) Phase contrast images merged with green fluorescence and histogram demonstrating significant differences (* p < 0.0001) in pcDNA-SH-SY5Y and TrkAIII SH-SY5Y percentage cell death at 24 h and 48 h, induced by 5 mM DTT.

Article Snippet: Polyclonal rabbit anti-human Y490 phosphorylated TrkA (9141), polyclonal rabbit anti-human Akt (9272), polyclonal rabbit anti-human phospho-Ser 473-Akt (4060), polyclonal rabbit anti-human Y674/675 (4621), antibody was from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Labeling, Control, Staining, Western Blot, Phospho-proteomics, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Molecular Characterization and Inhibition of a Novel Stress-Induced Mitochondrial Protecting Role for Misfolded TrkAIII in Human SH-SY5Y Neuroblastoma Cells

doi: 10.3390/ijms25105475

Figure Lengend Snippet: ( a ) Western blots demonstrating TrkAIII cleavage and phosphorylation in mitochondria (50 μg) from TrkAIII SH-SY5Y cells treated with 5 mM DTT alone for 6 h and in mitochondria from TrkAIII SH-SY5Y cells co-treated with 5 mM DTT and either HA-15 (20 μM) (DTT/HA); brefeldin A (5 mg/mL) (DTT/BfA) or W7 (60 μM) (DTT/W7) plus non-phosphorylated TrkAIII in mitochondria from untreated TrkAIII SH-SY5Y cells (Con) and lack of TrkA or phosphorylated TrkA immunoreactivity in mitochondria (50 μg) from untreated (Con) and DTT-treated (5 mm for 6 h) pcDNA-SH-SY5Y cells. ( b ) Line graphs demonstrating significant inhibition (*) of pcDNA-SH-SY5Y and TrkAIII SH-SY5Y proliferation by BfA (5 μg/mL) and W7 (60 μM) but not by HA-15 (20 μM) at 24 and 48 h (* p < 0.0001). ( c ) Phase contrast images merged with green fluorescence plus histograms demonstrating percentage pcDNA-SH-SY5Y and TrkAIII SH-SY5Y cell death induced by 5 mM DTT alone (DTT), DTT and either HA-15 (20 μM) (DTT/HA), BfA (5 μg/mL) (DTT/BfA) or W7 (60 μM) (DTT/W7), plus pcDNA-SH-SY5Y and TrkAIII SH-SY5Y cell death induced by W7 (60 μM) alone (W7) at 24 and 48 h (* p < 0.006).

Article Snippet: Polyclonal rabbit anti-human Y490 phosphorylated TrkA (9141), polyclonal rabbit anti-human Akt (9272), polyclonal rabbit anti-human phospho-Ser 473-Akt (4060), polyclonal rabbit anti-human Y674/675 (4621), antibody was from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Phospho-proteomics, Inhibition, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Molecular Characterization and Inhibition of a Novel Stress-Induced Mitochondrial Protecting Role for Misfolded TrkAIII in Human SH-SY5Y Neuroblastoma Cells

doi: 10.3390/ijms25105475

Figure Lengend Snippet: ( a ) Indirect IFs demonstrating increased overlapping (yellow/orange) immunoreactivity for TrkA (green) and MitoTracker-labeled mitochondria (red) (upper panels) and Y490 phosphorylated TrkAIII (pTrkAIII, green) and MitoTracker-labeled mitochondria (red) (lower panels) in DTT-treated (5 mM for 6 h) TrkAIII SH-SY5Y cells compared to untreated controls (control). DAPI stained nuclei are blue. (bar = 50 μm). ( b ) Western blots demonstrating TrkAIII cleavage and Y674/5 phosphorylation in mitochondria (50 mg) from DTT-treated (5 mM for 6 h) TrkAIII SH-SY5Y cells compared to untreated TrkAIII SH-SY5Y controls. ( c ) Western blots demonstrating increased PTPase oxidation (arrows) in mitochondria (50 mg) from DTT-treated TrkAIII SH-SY5Y cells (5 mM for 6 h) compared to untreated controls (Con). ( d ) Phase contrast images merged with green fluorescence and histogram demonstrating significant differences (* p < 0.0001) in pcDNA-SH-SY5Y and TrkAIII SH-SY5Y percentage cell death at 24 h and 48 h, induced by 5 mM DTT.

Article Snippet: Mouse monoclonal anti-human TrkA (B3, sc-7268), rabbit polyclonal anti-human XIAP (H-202), rabbit polyclonal anti-human α-tubulin (H-3000) and monoclonal mouse anti-human calmodulin (sc-137079) antibodies were from SantaCruz (Santa Cruz, CA, USA).

Techniques: Labeling, Control, Staining, Western Blot, Phospho-proteomics, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Molecular Characterization and Inhibition of a Novel Stress-Induced Mitochondrial Protecting Role for Misfolded TrkAIII in Human SH-SY5Y Neuroblastoma Cells

doi: 10.3390/ijms25105475

Figure Lengend Snippet: ( a ) Western blots demonstrating differences in TrkAIII immunoreactivity in untreated (CON) and DTT-treated (5 mM DTT for 1, 2 and 3 h) TrkAIII SH-SY5Y cell extracts (30 mg) under non-reducing and reducing conditions. ( b ) Co-immunoprecipitation Western blots demonstrating increased TrkAIII pulldown of Grp78 by anti-TrkA antibody in DTT-treated (5 mM for 6 h) TrkAIII SH-SY5Y cell extracts compared to untreated control extracts (CON) and similar levels of TrkAIII immunoprecipitated by anti-TrkA antibody but not by pre-immune mouse IgG in untreated (Con) and DTT-treated (5 mM for 6 h) TrkAIII SH-SY5Y cell extracts (500 μg), plus the absence of GRP78 or TrkA isoform pulldown by anti-TrkA antibody in pcDNA-SH-SY5Y cell extracts (500 μg). ( c ) Western blots of TrkAIII pulldown by calmodulin-conjugated Sepharose (CaM-Seph) in TrkAIII SH-SY5Y but not pcDNA-SH-SY5Y cell extracts (500 μg) in the presence of 150 mM CaCl 2 (left panel) plus increased TrkAIII pulldown by calmodulin-conjugated Sepharose (CaM-Seph) in TrkAIII SH-SY5Y cell extracts (500 μg) in the presence of 150 μM CaCl 2 compared to 5 mM EGTA (right panel), plus no TrkAIII pulldown by unconjugated Sepharose in TrkAIII SH-SY5Y cell extracts in the presence of 150 μM CaCl 2 or 5 mM EGTA (right panel). ( d ) Co-immunoprecipitation Western blots demonstrating enhanced pulldown of TrkAIII by anti-calmodulin antibody (anti-CaM) but not by pre-immune mouse IgG in DTT-treated (5 mM for 3 and 6 h) but not untreated (Con) TrkAIII SH-SY5Y extracts (500 μg) (upper left panels) and calmodulin pulldown by anti-calmodulin antibody (anti-CaM) but not pre-immune IgG in untreated (Con) and DTT-treated (DTT) TrkAIII SH-SY5Y cell extracts (500 μg) (upper right panels), plus no pulldown of TrkAIII by anti-calmodulin antibody in pcDNA-SH-SY5Y extracts (500 μg).

Article Snippet: Mouse monoclonal anti-human TrkA (B3, sc-7268), rabbit polyclonal anti-human XIAP (H-202), rabbit polyclonal anti-human α-tubulin (H-3000) and monoclonal mouse anti-human calmodulin (sc-137079) antibodies were from SantaCruz (Santa Cruz, CA, USA).

Techniques: Western Blot, Immunoprecipitation, Control

Journal: International Journal of Molecular Sciences

Article Title: Molecular Characterization and Inhibition of a Novel Stress-Induced Mitochondrial Protecting Role for Misfolded TrkAIII in Human SH-SY5Y Neuroblastoma Cells

doi: 10.3390/ijms25105475

Figure Lengend Snippet: ( a ) Western blots demonstrating TrkAIII cleavage and phosphorylation in mitochondria (50 μg) from TrkAIII SH-SY5Y cells treated with 5 mM DTT alone for 6 h and in mitochondria from TrkAIII SH-SY5Y cells co-treated with 5 mM DTT and either HA-15 (20 μM) (DTT/HA); brefeldin A (5 mg/mL) (DTT/BfA) or W7 (60 μM) (DTT/W7) plus non-phosphorylated TrkAIII in mitochondria from untreated TrkAIII SH-SY5Y cells (Con) and lack of TrkA or phosphorylated TrkA immunoreactivity in mitochondria (50 μg) from untreated (Con) and DTT-treated (5 mm for 6 h) pcDNA-SH-SY5Y cells. ( b ) Line graphs demonstrating significant inhibition (*) of pcDNA-SH-SY5Y and TrkAIII SH-SY5Y proliferation by BfA (5 μg/mL) and W7 (60 μM) but not by HA-15 (20 μM) at 24 and 48 h (* p < 0.0001). ( c ) Phase contrast images merged with green fluorescence plus histograms demonstrating percentage pcDNA-SH-SY5Y and TrkAIII SH-SY5Y cell death induced by 5 mM DTT alone (DTT), DTT and either HA-15 (20 μM) (DTT/HA), BfA (5 μg/mL) (DTT/BfA) or W7 (60 μM) (DTT/W7), plus pcDNA-SH-SY5Y and TrkAIII SH-SY5Y cell death induced by W7 (60 μM) alone (W7) at 24 and 48 h (* p < 0.006).

Article Snippet: Mouse monoclonal anti-human TrkA (B3, sc-7268), rabbit polyclonal anti-human XIAP (H-202), rabbit polyclonal anti-human α-tubulin (H-3000) and monoclonal mouse anti-human calmodulin (sc-137079) antibodies were from SantaCruz (Santa Cruz, CA, USA).

Techniques: Western Blot, Phospho-proteomics, Inhibition, Fluorescence

Journal: Scientific Reports

Article Title: Characterization of human iPSC-derived sensory neurons and their functional assessment using multi electrode array

doi: 10.1038/s41598-024-55602-8

Figure Lengend Snippet: Expression of sensory neuron related genes in hiPSC, hiPSC-derived sensory neurons and human DRG. Real-time PCR showed expression of ( a ) Peripherin, ( b ) Brn3a, ( c ) TRPV1, ( d ) TRPA1, ( e ) TRPM8, ( f ) Nav1.7, ( g ) Nav1.8, ( h ) Piezo2, ( i ) TRKA, ( j ) TRKB, ( k ) TRKC, ( l ) P2X3, ( m ) H1R, ( n ) MrgprX1, ( o ) CGRP, ( p )TAC1. The square marker, the circle marker and triangle marker indicate expression of genes in hiPSC, hiPSC-derived sensory neurons and human DRG respectively. Three different lot of hiPSC-derived sensory neurons were examined. The line marker represents the mean expression of genes in hiPSC-derived sensory neurons.

Article Snippet: Fixed samples were incubated with primary antibodies against TUBB3 (1:1000, Covance, PRB-435P), Brn3a (1:25, Millipore, MAB1585), Peripherin (1:200, Millipore, AB1530), TRPV1 (1:100, Invitrogen, PA1-748), TRPM8 (1:100, NOVUS, NBP1-97311), Nav1.7 (1:100, NOVUS, NBP2-12904), TRKA (1:80, R&D SYSTEMS, MAB1751R), TRKB (1:80, R&D SYSTEMS, MAB3971), TRKC (1:100, R&D SYSTEMS, AF373), Neurofilament 200 (1:1000, Sigma, N4142), Isolectin B4 (1:1000, Thermo Fisher Scientific, I21411) over night at 4 °C.

Techniques: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Marker

Journal: Scientific Reports

Article Title: Characterization of human iPSC-derived sensory neurons and their functional assessment using multi electrode array

doi: 10.1038/s41598-024-55602-8

Figure Lengend Snippet: Expression of sensory neuron related proteins in hiPSC-derived sensory neurons and their morphology. The cells are stained for ( a ) TUBB3, ( b ) Peripherin, ( c ) Brn3a, ( d ) TRPV1, ( e ) TRPM8, ( f ) Nav1.7, ( g ) TRKA, ( h ) TRKB, ( i ) TRKC, ( j ) NF200, ( k ) IB4. DAPI stain of nuclei is shown in blue. ( l ) Image of iPSC-derived sensory neurons which exhibit a bipolar (red arrowhead), pseudounipolar (yellow arrowhead), or multipolar morphology (green arrowhead). Scale bar represents 50 µm.

Article Snippet: Fixed samples were incubated with primary antibodies against TUBB3 (1:1000, Covance, PRB-435P), Brn3a (1:25, Millipore, MAB1585), Peripherin (1:200, Millipore, AB1530), TRPV1 (1:100, Invitrogen, PA1-748), TRPM8 (1:100, NOVUS, NBP1-97311), Nav1.7 (1:100, NOVUS, NBP2-12904), TRKA (1:80, R&D SYSTEMS, MAB1751R), TRKB (1:80, R&D SYSTEMS, MAB3971), TRKC (1:100, R&D SYSTEMS, AF373), Neurofilament 200 (1:1000, Sigma, N4142), Isolectin B4 (1:1000, Thermo Fisher Scientific, I21411) over night at 4 °C.

Techniques: Expressing, Derivative Assay, Staining