polyclonal rabbit antibody ps6 ser 235 236  (Cell Signaling Technology Inc)

Journal: Frontiers in Cell and Developmental Biology

Article Title: miR-514a promotes neuronal development in human iPSC-derived neurons

doi: 10.3389/fcell.2023.1096463

Figure Lengend Snippet: miR-514a upregulates mTOR signaling pathway in human iPSC-derived neurons. Neurons were infected with lentiviruses expressing miR-514a and cultured for 7 days. (A–C) Representative images of iPSC-derived neurons stained with anti-pS6 (red) antibody and Hoechst 33,258 (magenta) at 7 DIV. Scale bars, 100 µm. (D–F) pS6 positive neurons derived from three independent iPSC line, respectively, were quantified. mTOR activity was markedly increased in neurons expressed miR-514a. n = 3 independent experiments, at least 200 neurons were analyzed in each experiment. (G) Western blotting analysis showing the level of pS6 in cultured neurons at 7 DIV. (H) Quantification of pS6 level. n = 3 independent experiments. *** p < 0.001, ** p < 0.01, * p < 0.05. Abbreviations: pS6, phospho-S6 ribosomal protein.

Article Snippet: The following primary antibodies were used for western blotting: polyclonal rabbit antibody pS6 Ser 235/236 (1:500; Cell Signaling Technology) and monoclonal rabbit antibody β-actin (1:500; Cell Signaling Technology).

Techniques: Derivative Assay, Infection, Expressing, Cell Culture, Staining, Activity Assay, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: miR-514a promotes neuronal development in human iPSC-derived neurons

doi: 10.3389/fcell.2023.1096463

Figure Lengend Snippet: Blocking miR-514a using its sponges causes inhibition of neuronal development in iPSC-derived neurons. Neurons were infected with lentiviruses expressing miR-514a-5p sponge, miR-514a-3p sponge or scrambled shRNA as control and cultured for 7 days. (A) Diagrams of lentiviral vector constructs expressing sponges against miR-514a-5p and miR-514a-3p. The sponges were expressed under the U6 RNA polymerase Ⅲ promotor, and EGFP is expressed under the CMV promotor. (B) Representative images of iPSC-derived neurons (201B7) stained with anti-MAP2 (blue) antibody at 7 DIV. Scale bars, 50 µm. (C) Representative images of iPSC-derived neurons (201B7) stained with anti-MAP2 (blue) antibody at 7 DIV. Scale bars, 25 µm. (D) Representative images of iPSC-derived neurons (201B7) stained with anti-pS6 (red) antibody and Hoechst 33,258 (magenta) at 7 DIV. Scale bars, 100 µm. (E) Quantitative analysis of total dendrite length. n = 3 independent experiments, at least 100 neurons were analyzed in each experiment. (F) Quantitative analysis of neuronal soma size. n = 3 independent experiments, at least 100 neurons were analyzed in each experiment. (G) Quantitative analysis of pS6 positive cells. n = 3 independent experiments, at least 200 cells were analyzed in each experiment. n = 3 independent experiments. *** p < 0.001, ** p < 0.01, * p < 0.05. Abbreviations: CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; MAP2, microtubule-associated protein 2; pS6, phospho-S6 ribosomal protein; n. s., not significant.

Article Snippet: The following primary antibodies were used for western blotting: polyclonal rabbit antibody pS6 Ser 235/236 (1:500; Cell Signaling Technology) and monoclonal rabbit antibody β-actin (1:500; Cell Signaling Technology).

Techniques: Blocking Assay, Inhibition, Derivative Assay, Infection, Expressing, shRNA, Control, Cell Culture, Plasmid Preparation, Construct, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: miR-514a promotes neuronal development in human iPSC-derived neurons

doi: 10.3389/fcell.2023.1096463

Figure Lengend Snippet: Variation in miR-514a sequences among primate genomes affects the expression level of mature miR-514a. (A) Comparison of sequence of precursor-miR-514a across species. (B) Schematic representation of the mutation in pre-miR-514a-3. (the nucleotide position in the mutation is indicated with arrow). (C) Expression levels of mature miR-514a were measured using qRT-PCR in 201B7 iPSC-derived neurons infected with lentiviruses expressing native miR-514a, miR-514a mutant or scrambled shRNA as control. n = 3 independent experiments. (D) Representative images of iPSC-derived neurons stained with anti-MAP2 (blue) antibody at 7 DIV. Scale bars, 50 µm. (E) Representative images of iPSC-derived neurons stained with anti-MAP2 (blue) antibody at 7 DIV. Scale bars, 25 µm. (F) Representative images of iPSC-derived neurons stained with anti-pS6 (red) antibody and Hoechst 33,258 (magenta) at 7 DIV. Scale bars, 100 µm. (G) Quantitative analysis of total dendrite length. n = 3 independent experiments, at least 100 neurons were analyzed in each experiment. (H) Quantitative analysis of neuronal soma size. n = 3 independent experiments, at least 100 neurons were analyzed in each experiment. (I) Quantitative analysis of pS6 positive cells. n = 3 independent experiments, at least 200 cells were analyzed in each experiment. ** p < 0.01, * p < 0.05. Abbreviations: MAP2, microtubule-associated protein 2; pS6, phospho-S6 ribosomal protein; n. s., not significant.

Article Snippet: The following primary antibodies were used for western blotting: polyclonal rabbit antibody pS6 Ser 235/236 (1:500; Cell Signaling Technology) and monoclonal rabbit antibody β-actin (1:500; Cell Signaling Technology).

Techniques: Expressing, Comparison, Sequencing, Mutagenesis, Quantitative RT-PCR, Derivative Assay, Infection, shRNA, Control, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: miR-514a promotes neuronal development in human iPSC-derived neurons

doi: 10.3389/fcell.2023.1096463

Figure Lengend Snippet: miR-514a upregulates mTOR signaling pathway in human iPSC-derived neurons. Neurons were infected with lentiviruses expressing miR-514a and cultured for 7 days. (A–C) Representative images of iPSC-derived neurons stained with anti-pS6 (red) antibody and Hoechst 33,258 (magenta) at 7 DIV. Scale bars, 100 µm. (D–F) pS6 positive neurons derived from three independent iPSC line, respectively, were quantified. mTOR activity was markedly increased in neurons expressed miR-514a. n = 3 independent experiments, at least 200 neurons were analyzed in each experiment. (G) Western blotting analysis showing the level of pS6 in cultured neurons at 7 DIV. (H) Quantification of pS6 level. n = 3 independent experiments. *** p < 0.001, ** p < 0.01, * p < 0.05. Abbreviations: pS6, phospho-S6 ribosomal protein.

Article Snippet: The following primary antibodies were used for immunocytochemistry: monoclonal mouse antibody β-III tubulin (Tuj-1) (1:1,000; Sigma), polyclonal guinea pig antibody microtubule-associated protein 2 (MAP2) (1:1,000; Synaptic Systems, Goettingen, Germany) and polyclonal rabbit antibody phospho-S6 ribosomal protein (pS6) Ser 235/236 (1:1,000; Cell Signaling Technology, Danvers, MA, United States).

Techniques: Derivative Assay, Infection, Expressing, Cell Culture, Staining, Activity Assay, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: miR-514a promotes neuronal development in human iPSC-derived neurons

doi: 10.3389/fcell.2023.1096463

Figure Lengend Snippet: Blocking miR-514a using its sponges causes inhibition of neuronal development in iPSC-derived neurons. Neurons were infected with lentiviruses expressing miR-514a-5p sponge, miR-514a-3p sponge or scrambled shRNA as control and cultured for 7 days. (A) Diagrams of lentiviral vector constructs expressing sponges against miR-514a-5p and miR-514a-3p. The sponges were expressed under the U6 RNA polymerase Ⅲ promotor, and EGFP is expressed under the CMV promotor. (B) Representative images of iPSC-derived neurons (201B7) stained with anti-MAP2 (blue) antibody at 7 DIV. Scale bars, 50 µm. (C) Representative images of iPSC-derived neurons (201B7) stained with anti-MAP2 (blue) antibody at 7 DIV. Scale bars, 25 µm. (D) Representative images of iPSC-derived neurons (201B7) stained with anti-pS6 (red) antibody and Hoechst 33,258 (magenta) at 7 DIV. Scale bars, 100 µm. (E) Quantitative analysis of total dendrite length. n = 3 independent experiments, at least 100 neurons were analyzed in each experiment. (F) Quantitative analysis of neuronal soma size. n = 3 independent experiments, at least 100 neurons were analyzed in each experiment. (G) Quantitative analysis of pS6 positive cells. n = 3 independent experiments, at least 200 cells were analyzed in each experiment. n = 3 independent experiments. *** p < 0.001, ** p < 0.01, * p < 0.05. Abbreviations: CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; MAP2, microtubule-associated protein 2; pS6, phospho-S6 ribosomal protein; n. s., not significant.

Article Snippet: The following primary antibodies were used for immunocytochemistry: monoclonal mouse antibody β-III tubulin (Tuj-1) (1:1,000; Sigma), polyclonal guinea pig antibody microtubule-associated protein 2 (MAP2) (1:1,000; Synaptic Systems, Goettingen, Germany) and polyclonal rabbit antibody phospho-S6 ribosomal protein (pS6) Ser 235/236 (1:1,000; Cell Signaling Technology, Danvers, MA, United States).

Techniques: Blocking Assay, Inhibition, Derivative Assay, Infection, Expressing, shRNA, Control, Cell Culture, Plasmid Preparation, Construct, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: miR-514a promotes neuronal development in human iPSC-derived neurons

doi: 10.3389/fcell.2023.1096463

Figure Lengend Snippet: Variation in miR-514a sequences among primate genomes affects the expression level of mature miR-514a. (A) Comparison of sequence of precursor-miR-514a across species. (B) Schematic representation of the mutation in pre-miR-514a-3. (the nucleotide position in the mutation is indicated with arrow). (C) Expression levels of mature miR-514a were measured using qRT-PCR in 201B7 iPSC-derived neurons infected with lentiviruses expressing native miR-514a, miR-514a mutant or scrambled shRNA as control. n = 3 independent experiments. (D) Representative images of iPSC-derived neurons stained with anti-MAP2 (blue) antibody at 7 DIV. Scale bars, 50 µm. (E) Representative images of iPSC-derived neurons stained with anti-MAP2 (blue) antibody at 7 DIV. Scale bars, 25 µm. (F) Representative images of iPSC-derived neurons stained with anti-pS6 (red) antibody and Hoechst 33,258 (magenta) at 7 DIV. Scale bars, 100 µm. (G) Quantitative analysis of total dendrite length. n = 3 independent experiments, at least 100 neurons were analyzed in each experiment. (H) Quantitative analysis of neuronal soma size. n = 3 independent experiments, at least 100 neurons were analyzed in each experiment. (I) Quantitative analysis of pS6 positive cells. n = 3 independent experiments, at least 200 cells were analyzed in each experiment. ** p < 0.01, * p < 0.05. Abbreviations: MAP2, microtubule-associated protein 2; pS6, phospho-S6 ribosomal protein; n. s., not significant.

Article Snippet: The following primary antibodies were used for immunocytochemistry: monoclonal mouse antibody β-III tubulin (Tuj-1) (1:1,000; Sigma), polyclonal guinea pig antibody microtubule-associated protein 2 (MAP2) (1:1,000; Synaptic Systems, Goettingen, Germany) and polyclonal rabbit antibody phospho-S6 ribosomal protein (pS6) Ser 235/236 (1:1,000; Cell Signaling Technology, Danvers, MA, United States).

Techniques: Expressing, Comparison, Sequencing, Mutagenesis, Quantitative RT-PCR, Derivative Assay, Infection, shRNA, Control, Staining