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Schematic of the generation of <t>IRS2</t> knock-in cells. A , targeted integration of AID (mIAA7), FLAG (3XF), and mNeon-green (mNG) tags with linker (L) into the N-terminus of IRS2 and integration of At AFB2 and the PuroR cassette into the AAVS1 safe harbor locus in homozygously tagged clones by selection with puromycin. LHA, left homology arm; RHA, right homology arm. B , PCR genotyping strategy for selecting homozygously tagged DFN-IRS2 . First-round primers complementary to the modified loci were used to amplify wild-type alleles (523 bp) and knock-in alleles (1663 bp). Second-round primers complementary to IRS2 outside the donor plasmid homology arms were used in long-distance PCR for wild-type alleles (2260 bp) and knock-in alleles (3400 bp). Indicated amplicon sizes confirm the homozygous integration of the tags.
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Schematic of the generation of <t>IRS2</t> knock-in cells. A , targeted integration of AID (mIAA7), FLAG (3XF), and mNeon-green (mNG) tags with linker (L) into the N-terminus of IRS2 and integration of At AFB2 and the PuroR cassette into the AAVS1 safe harbor locus in homozygously tagged clones by selection with puromycin. LHA, left homology arm; RHA, right homology arm. B , PCR genotyping strategy for selecting homozygously tagged DFN-IRS2 . First-round primers complementary to the modified loci were used to amplify wild-type alleles (523 bp) and knock-in alleles (1663 bp). Second-round primers complementary to IRS2 outside the donor plasmid homology arms were used in long-distance PCR for wild-type alleles (2260 bp) and knock-in alleles (3400 bp). Indicated amplicon sizes confirm the homozygous integration of the tags.
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Image Search Results


Schematic of the generation of IRS2 knock-in cells. A , targeted integration of AID (mIAA7), FLAG (3XF), and mNeon-green (mNG) tags with linker (L) into the N-terminus of IRS2 and integration of At AFB2 and the PuroR cassette into the AAVS1 safe harbor locus in homozygously tagged clones by selection with puromycin. LHA, left homology arm; RHA, right homology arm. B , PCR genotyping strategy for selecting homozygously tagged DFN-IRS2 . First-round primers complementary to the modified loci were used to amplify wild-type alleles (523 bp) and knock-in alleles (1663 bp). Second-round primers complementary to IRS2 outside the donor plasmid homology arms were used in long-distance PCR for wild-type alleles (2260 bp) and knock-in alleles (3400 bp). Indicated amplicon sizes confirm the homozygous integration of the tags.

Journal: The Journal of Biological Chemistry

Article Title: Fluorescent tagging of endogenous IRS2 with an auxin-dependent degron to assess dynamic intracellular localization and function

doi: 10.1016/j.jbc.2024.107796

Figure Lengend Snippet: Schematic of the generation of IRS2 knock-in cells. A , targeted integration of AID (mIAA7), FLAG (3XF), and mNeon-green (mNG) tags with linker (L) into the N-terminus of IRS2 and integration of At AFB2 and the PuroR cassette into the AAVS1 safe harbor locus in homozygously tagged clones by selection with puromycin. LHA, left homology arm; RHA, right homology arm. B , PCR genotyping strategy for selecting homozygously tagged DFN-IRS2 . First-round primers complementary to the modified loci were used to amplify wild-type alleles (523 bp) and knock-in alleles (1663 bp). Second-round primers complementary to IRS2 outside the donor plasmid homology arms were used in long-distance PCR for wild-type alleles (2260 bp) and knock-in alleles (3400 bp). Indicated amplicon sizes confirm the homozygous integration of the tags.

Article Snippet: Primary antibodies: rabbit IRS2 (#4502; Cell Signaling Technology), rabbit mNeon-green (#55074; Cell Signaling Technology), mouse GAPDH (#sc-32233; Santa Cruz), mouse pTyr (sc-7020; Santa Cruz), rabbit p85 (#4292; Cell Signaling Technology), rabbit AKT (#9272; Cell Signaling Technology), rabbit pS473 AKT (#9271; Cell Signaling Technology), mouse FLAG (F1804; Sigma), rabbit HA (#3724; Cell Signaling Technology), mouse Lamin A/C (#sc-376248; Santa Cruz), mouse Actin (# MA5-11869; Thermo Fisher Scientific).

Techniques: Knock-In, Clone Assay, Selection, Modification, Plasmid Preparation, Amplification

Characterization of DFN-IRS2 protein expression and function. A , cell extracts from parental (WT) and DFN (KI) SUM-159 and MDA-MB-231 cells were immunoblotted with antibodies that detect IRS2, mNeon-green and FLAG. B , fluorescence imaging of parental (WT) and DFN-IRS2 (KI) SUM-159 and MDA-MB-231 cells. Scale bars = 25 μm (SUM-159) or 10 μm (MDA-MB-231). C , flow cytometric analysis of parental (WT) and DFN-IRS2 (KI) SUM-159 and MDA-MB-231 cells. D and E , parental (WT) and DFN-IRS2 (KI) SUM-159 and MDA-MB-231 cells were serum starved and stimulated with IGF-1 (50 ng/ml) for 10 min. Aliquots of cell extracts that contained equivalent amounts of total protein were immunoprecipitated with IRS2-specific antibodies and the immune complexes were immunoblotted with antibodies that recognize either IRS2, phosphotyrosine (pTyr), or p85. Aliquots of cell extracts that contained equivalent amounts of total protein (WCL) were immunoblotted with the indicated antibodies.

Journal: The Journal of Biological Chemistry

Article Title: Fluorescent tagging of endogenous IRS2 with an auxin-dependent degron to assess dynamic intracellular localization and function

doi: 10.1016/j.jbc.2024.107796

Figure Lengend Snippet: Characterization of DFN-IRS2 protein expression and function. A , cell extracts from parental (WT) and DFN (KI) SUM-159 and MDA-MB-231 cells were immunoblotted with antibodies that detect IRS2, mNeon-green and FLAG. B , fluorescence imaging of parental (WT) and DFN-IRS2 (KI) SUM-159 and MDA-MB-231 cells. Scale bars = 25 μm (SUM-159) or 10 μm (MDA-MB-231). C , flow cytometric analysis of parental (WT) and DFN-IRS2 (KI) SUM-159 and MDA-MB-231 cells. D and E , parental (WT) and DFN-IRS2 (KI) SUM-159 and MDA-MB-231 cells were serum starved and stimulated with IGF-1 (50 ng/ml) for 10 min. Aliquots of cell extracts that contained equivalent amounts of total protein were immunoprecipitated with IRS2-specific antibodies and the immune complexes were immunoblotted with antibodies that recognize either IRS2, phosphotyrosine (pTyr), or p85. Aliquots of cell extracts that contained equivalent amounts of total protein (WCL) were immunoblotted with the indicated antibodies.

Article Snippet: Primary antibodies: rabbit IRS2 (#4502; Cell Signaling Technology), rabbit mNeon-green (#55074; Cell Signaling Technology), mouse GAPDH (#sc-32233; Santa Cruz), mouse pTyr (sc-7020; Santa Cruz), rabbit p85 (#4292; Cell Signaling Technology), rabbit AKT (#9272; Cell Signaling Technology), rabbit pS473 AKT (#9271; Cell Signaling Technology), mouse FLAG (F1804; Sigma), rabbit HA (#3724; Cell Signaling Technology), mouse Lamin A/C (#sc-376248; Santa Cruz), mouse Actin (# MA5-11869; Thermo Fisher Scientific).

Techniques: Expressing, Fluorescence, Imaging, Immunoprecipitation

Analysis of DFN-IRS2 intracellular localization by live imaging. A , DFN-IRS2 (KI) SUM-159, and MDA-MB-231 cells were analyzed by live fluorescence imaging in a complete culture medium or culture medium lacking FBS (serum starve). The data shown in the graphs on the right represent the mean ± S.D. of the nuclear/cytoplasmic ratio of DFN-IRS2 (n = 24) from a representative experiment. Scale bars = 10 μm. B , DFN-IRS2 KI MDA-MB-231 cells were imaged for 8 h in complete culture medium or culture medium lacking FBS (serum starve) for 8 h. Top row, cells began in full culture medium and were serum starved for 8 h. Bottom row, cells began under serum-starved conditions and were incubated in complete culture medium for 8 h. The images shown capture the start and end of the imaging period. The data shown in the graphs represent the nuclear/cytoplasmic ratio of DFN-IRS2 in individual cells at the beginning and end of the imaging (n = 10). Colored arrows track individual cells over time. Scale bars = 10 μm. C , Parental MDA-MB-231 cells were grown in a normal culture medium or culture medium lacking FBS (SS) and extracted to isolate cytosolic and nuclear fractions. Aliquots of cell extracts that contained equivalent amounts of total protein were immunoblotted with the indicated antibodies. The data shown represent the mean ± S.D. of the nuclear/cytoplasmic ratio from five independent experiments. D , parental SUM-159 cells were grown in a normal culture medium in the absence (−) or presence of BKM120 or vehicle (DMSO) and extracted to isolate cytosolic and nuclear fractions. Aliquots of cell extracts that contained equivalent amounts of total protein were immunoblotted with the indicated antibodies. The data shown represent the mean ± S.D. of the nuclear/cytoplasmic ratio from four independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Journal: The Journal of Biological Chemistry

Article Title: Fluorescent tagging of endogenous IRS2 with an auxin-dependent degron to assess dynamic intracellular localization and function

doi: 10.1016/j.jbc.2024.107796

Figure Lengend Snippet: Analysis of DFN-IRS2 intracellular localization by live imaging. A , DFN-IRS2 (KI) SUM-159, and MDA-MB-231 cells were analyzed by live fluorescence imaging in a complete culture medium or culture medium lacking FBS (serum starve). The data shown in the graphs on the right represent the mean ± S.D. of the nuclear/cytoplasmic ratio of DFN-IRS2 (n = 24) from a representative experiment. Scale bars = 10 μm. B , DFN-IRS2 KI MDA-MB-231 cells were imaged for 8 h in complete culture medium or culture medium lacking FBS (serum starve) for 8 h. Top row, cells began in full culture medium and were serum starved for 8 h. Bottom row, cells began under serum-starved conditions and were incubated in complete culture medium for 8 h. The images shown capture the start and end of the imaging period. The data shown in the graphs represent the nuclear/cytoplasmic ratio of DFN-IRS2 in individual cells at the beginning and end of the imaging (n = 10). Colored arrows track individual cells over time. Scale bars = 10 μm. C , Parental MDA-MB-231 cells were grown in a normal culture medium or culture medium lacking FBS (SS) and extracted to isolate cytosolic and nuclear fractions. Aliquots of cell extracts that contained equivalent amounts of total protein were immunoblotted with the indicated antibodies. The data shown represent the mean ± S.D. of the nuclear/cytoplasmic ratio from five independent experiments. D , parental SUM-159 cells were grown in a normal culture medium in the absence (−) or presence of BKM120 or vehicle (DMSO) and extracted to isolate cytosolic and nuclear fractions. Aliquots of cell extracts that contained equivalent amounts of total protein were immunoblotted with the indicated antibodies. The data shown represent the mean ± S.D. of the nuclear/cytoplasmic ratio from four independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Primary antibodies: rabbit IRS2 (#4502; Cell Signaling Technology), rabbit mNeon-green (#55074; Cell Signaling Technology), mouse GAPDH (#sc-32233; Santa Cruz), mouse pTyr (sc-7020; Santa Cruz), rabbit p85 (#4292; Cell Signaling Technology), rabbit AKT (#9272; Cell Signaling Technology), rabbit pS473 AKT (#9271; Cell Signaling Technology), mouse FLAG (F1804; Sigma), rabbit HA (#3724; Cell Signaling Technology), mouse Lamin A/C (#sc-376248; Santa Cruz), mouse Actin (# MA5-11869; Thermo Fisher Scientific).

Techniques: Imaging, Fluorescence, Incubation

Regulation of IRS2 intracellular localization. A , schematic of WT-IRS2 and IRS2-deletion mutants showing a putative nuclear export signal (NES). B , DFN-IRS2 (KI) MDA-MB-231 cells were imaged in a complete culture medium in the presence or absence of Leptomycin B over 8 h, and images from specific time points are shown. The data in the graphs represent the nuclear/cytoplasmic ratio of DFN-IRS2 at each time point in individual cells (n = 7) from a representative experiment. Colored arrows track individual cells over time. Scale bars = 10 μm. C , MDA-MB-231 DFN-IRS2 KI cells were imaged in a complete culture medium in the presence or absence of Leptomycin B for 8 h. Representative images from 0, 4, and 8 h are shown. The data in the graphs represent the mean ± S.D. of the nuclear/cytoplasmic ratio of DFN-IRS2 at each time point (n = 17). Scale bars = 10 μm. D , SUM-159 cells transfected with HA-tagged WT-IRS2 and IRS2 deletion mutants (shown in A) were fixed and stained with HA antibodies (green) and DAPI (blue). The data shown in the graph represent the mean ± S.D. of IRS2 expression in the nucleus from a representative experiment performed three independent times (n = 40–43 cells). Scale bars = 50 μm. Top right, immunoblot of exogenous IRS2 expression. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Journal: The Journal of Biological Chemistry

Article Title: Fluorescent tagging of endogenous IRS2 with an auxin-dependent degron to assess dynamic intracellular localization and function

doi: 10.1016/j.jbc.2024.107796

Figure Lengend Snippet: Regulation of IRS2 intracellular localization. A , schematic of WT-IRS2 and IRS2-deletion mutants showing a putative nuclear export signal (NES). B , DFN-IRS2 (KI) MDA-MB-231 cells were imaged in a complete culture medium in the presence or absence of Leptomycin B over 8 h, and images from specific time points are shown. The data in the graphs represent the nuclear/cytoplasmic ratio of DFN-IRS2 at each time point in individual cells (n = 7) from a representative experiment. Colored arrows track individual cells over time. Scale bars = 10 μm. C , MDA-MB-231 DFN-IRS2 KI cells were imaged in a complete culture medium in the presence or absence of Leptomycin B for 8 h. Representative images from 0, 4, and 8 h are shown. The data in the graphs represent the mean ± S.D. of the nuclear/cytoplasmic ratio of DFN-IRS2 at each time point (n = 17). Scale bars = 10 μm. D , SUM-159 cells transfected with HA-tagged WT-IRS2 and IRS2 deletion mutants (shown in A) were fixed and stained with HA antibodies (green) and DAPI (blue). The data shown in the graph represent the mean ± S.D. of IRS2 expression in the nucleus from a representative experiment performed three independent times (n = 40–43 cells). Scale bars = 50 μm. Top right, immunoblot of exogenous IRS2 expression. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: Primary antibodies: rabbit IRS2 (#4502; Cell Signaling Technology), rabbit mNeon-green (#55074; Cell Signaling Technology), mouse GAPDH (#sc-32233; Santa Cruz), mouse pTyr (sc-7020; Santa Cruz), rabbit p85 (#4292; Cell Signaling Technology), rabbit AKT (#9272; Cell Signaling Technology), rabbit pS473 AKT (#9271; Cell Signaling Technology), mouse FLAG (F1804; Sigma), rabbit HA (#3724; Cell Signaling Technology), mouse Lamin A/C (#sc-376248; Santa Cruz), mouse Actin (# MA5-11869; Thermo Fisher Scientific).

Techniques: Transfection, Staining, Expressing, Western Blot

Auxin-mediated degradation of DFN-IRS2. A , DFN-IRS2 (KI) cells were treated with or without 3-IAA (0.5 mM) for the indicated time periods and cell extracts were immunoblotted with IRS2 antibodies. The data shown in the graphs represent the mean ± S.D. of three independent experiments. B , DFN-IRS2 (KI) cells were treated with or without 0.5 mM 3-IAA for 6 h. The cells were then washed and allowed to recover for the indicated time periods in complete culture medium. The data shown in the graphs represent the mean ± S.D. of three independent experiments. C , parental (WT) and DFN-IRS2 (KI) SUM-159 cells were treated with 0.5 mM 3-IAA and assayed for invasion using Matrigel Transwell assays. The data shown represent the mean ± S.D. of invasion relative to untreated control cells from two independent experiments. Expression of DFN-IRS2 at the beginning and end of the assay is shown in the immunoblot. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Journal: The Journal of Biological Chemistry

Article Title: Fluorescent tagging of endogenous IRS2 with an auxin-dependent degron to assess dynamic intracellular localization and function

doi: 10.1016/j.jbc.2024.107796

Figure Lengend Snippet: Auxin-mediated degradation of DFN-IRS2. A , DFN-IRS2 (KI) cells were treated with or without 3-IAA (0.5 mM) for the indicated time periods and cell extracts were immunoblotted with IRS2 antibodies. The data shown in the graphs represent the mean ± S.D. of three independent experiments. B , DFN-IRS2 (KI) cells were treated with or without 0.5 mM 3-IAA for 6 h. The cells were then washed and allowed to recover for the indicated time periods in complete culture medium. The data shown in the graphs represent the mean ± S.D. of three independent experiments. C , parental (WT) and DFN-IRS2 (KI) SUM-159 cells were treated with 0.5 mM 3-IAA and assayed for invasion using Matrigel Transwell assays. The data shown represent the mean ± S.D. of invasion relative to untreated control cells from two independent experiments. Expression of DFN-IRS2 at the beginning and end of the assay is shown in the immunoblot. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Primary antibodies: rabbit IRS2 (#4502; Cell Signaling Technology), rabbit mNeon-green (#55074; Cell Signaling Technology), mouse GAPDH (#sc-32233; Santa Cruz), mouse pTyr (sc-7020; Santa Cruz), rabbit p85 (#4292; Cell Signaling Technology), rabbit AKT (#9272; Cell Signaling Technology), rabbit pS473 AKT (#9271; Cell Signaling Technology), mouse FLAG (F1804; Sigma), rabbit HA (#3724; Cell Signaling Technology), mouse Lamin A/C (#sc-376248; Santa Cruz), mouse Actin (# MA5-11869; Thermo Fisher Scientific).

Techniques: Control, Expressing, Western Blot

gRNA, Primer and Donor sequences

Journal: The Journal of Biological Chemistry

Article Title: Fluorescent tagging of endogenous IRS2 with an auxin-dependent degron to assess dynamic intracellular localization and function

doi: 10.1016/j.jbc.2024.107796

Figure Lengend Snippet: gRNA, Primer and Donor sequences

Article Snippet: Primary antibodies: rabbit IRS2 (#4502; Cell Signaling Technology), rabbit mNeon-green (#55074; Cell Signaling Technology), mouse GAPDH (#sc-32233; Santa Cruz), mouse pTyr (sc-7020; Santa Cruz), rabbit p85 (#4292; Cell Signaling Technology), rabbit AKT (#9272; Cell Signaling Technology), rabbit pS473 AKT (#9271; Cell Signaling Technology), mouse FLAG (F1804; Sigma), rabbit HA (#3724; Cell Signaling Technology), mouse Lamin A/C (#sc-376248; Santa Cruz), mouse Actin (# MA5-11869; Thermo Fisher Scientific).

Techniques: Sequencing