Journal: bioRxiv

Article Title: Proteome-wide multi-omics profiling of osteosarcoma transcription factor networks

doi: 10.64898/2026.03.29.714917

Figure Lengend Snippet: A) Schema of the concatenated tandem array of the consensus transcription factor response elements (catTFREs) workflow utilising two plasmids inserts C1 (designed with approximately 110 TF binding sites (TFBS) in duplicates with linkers) and C4 (reduced TFBS). Detailed TFBS are provided in Table S1A and B. Inserts were linearized by restriction enzyme digestion and biotinylated by Klenow enzyme. Biotinylated C1 and C4 were immobilized on streptavidin coated magnetic beads and incubated with nuclear extracts (NE) for detection of DNA binding proteins by mass spectrometry (MS). B) MS spectral counts of proteins enriched from NEs or by TFRE C1 and C4 pulldown. Four replicates with total count of proteins (left) and TFs only (right) from HOS (purple), U2OS (red) and MG63 (blue). All source data are provided in Table S1C-F. C) Heatmap representing the Pearson correlation of spectral counts by samples from HOS, U2OS and MG63. Scale represents low correlation in white to self-correlations as red-brown. D) Clustering of proteins by Weighted Gene Co-expression Network Analysis from MS spectral counts of NE and C1/C4 in HOS, MG63 and U2OS. Clusters I to X with the number of proteins represented by Pearson correlation across all conditions. Proteins are listed in Table S1G.

Article Snippet: The restriction digestion reactions were precipitated and biotinylated by biotinylated dUTP and dATP (Roche) using a Klenow Fragment (New England Biolabs).

Techniques: Binding Assay, Magnetic Beads, Incubation, DNA Binding Assay, Mass Spectrometry, Expressing

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 2. Neuronal activity bidirectionally modulates the phosphorylation state of Shank3. (A) The experiment protocol for extraction of Shank3 from rat cultured neocortical neurons for further quantitative mass spectrometry (MS) or Western blot analyses. (B) Volcano plot of quantitative MS data showing Shank3 residues that were differentially phosphorylated in tetrodotoxin (TTX)-treated samples compared to untreated controls. The log2 values of fold changes, if below zero, indicated hypophosphorylation (paired t-test: S1586, adjusted p=0.034142, S1614/5, 0.014444). (C) Top: diagram showing the location of S1586 and S1615 within the rat Shank3 protein. Functional domains: ANK = ankyrin repeat; SH3 = SRC homology 3; PDZ = PSD-95/Disc Large/ZO-1; Pro-rich = proline rich; SAM = sterile alpha motif. Bottom: homology comparison of sequences flanking rat S1586 and S1615 (matching mouse S1539) across species (human Shank3: NP_001358973.1; rat Shank3: NP_067708.2; mouse Shank3: UniprotKB: Q4ACU6.3). Phosphosites of interest are labeled in red; the only residue not conserved is shown in blue. (D, E) Representative Western blot using an antibody specific for phosphorylated S1615, showing changes in Shank3 phosphorylation after 10 min (D) or 24 hr (E) treatment with TTX or picrotoxin (PTX). (F) Quantification of the fold change of Shank3 S1615 phosphorylation in (D). Dashed line indicates the baseline untreated control (one-sample t-test: TTX, ***p=0.0005, PTX, **p=0.0035, n = 5 and 10 biological replicates, respectively). (G) Quantification of the fold change of Shank3 S1615 phosphorylation in (E) (one-sample t-test: TTX, ****p<0.0001, PTX, p = 0.6336, n = 7 and 7 biological replicates, respectively). Solid colored horizontal

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Activity Assay, Phospho-proteomics, Extraction, Cell Culture, Mass Spectrometry, Western Blot, Functional Assay, Sterility, Comparison, Labeling, Residue, Control

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 3. Phosphorylation state modulates activity-dependent changes in the synaptic enrichment of Shank3. (A) Representative images of synaptic puncta colocalized with surface GluA2 (sGluA2) and Shank3 in neuron dendrites ± tetrodotoxin (TTX) (scale bar = 5 µm). (B) Quantification of synaptic sGluA2 intensity changes induced by scaling up and down protocols (number of neurons: untreated, n = 77, TTX, n = 40, picrotoxin [PTX], n = 29; Kruskal–Wallis test with post-hoc Dunn’s multiple comparison tests: Un vs. TTX, **p=0.0034, Un vs. PTX, *p=0.0408, TTX vs. PTX, ****p<0.0001). (C) Quantification of synaptic Shank3 intensity during scaling up and down protocols (Kruskal–Wallis test with post-hoc Dunn’s tests: Un vs. TTX, *p=0.0155, Un vs. PTX, *p=0.0205, TTX vs. PTX, ****p<0.0001). (D) Representative images of synaptic localization of wild-type Shank3 and Shank3 phospho-mutants (scale bar = 5 µm). (E) Quantification of synaptic intensity of Shank3 phospho-mutants (number of neurons: WT, n = 33, AA, n = 30, DD, n = 24; Kruskal–Wallis test with post-hoc Dunn’s tests: WT vs. AA, p>0.9999, WT vs. DD, *p=0.0395, AA vs. DD, **p=0.0039). (F) Quantification of the density of synaptic puncta containing Shank3 phospho-mutants (number of neurons: WT, n = 32, AA, n = 30, DD, n = 24; Kruskal–Wallis test: p=0.2814). For imaging experiments here and below, each data point represents a single pyramidal neuron, and data were collected from at least four independent experiments. Also see Figure 3—source data 1.

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Phospho-proteomics, Activity Assay, Comparison, Imaging

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 4. Increased PP2A activity maintains tetrodotoxin (TTX)-induced Shank3 hypophosphorylation. (A) Diagram showing the potential roles of kinases and phosphatases in regulating activity-dependent Shank3 phosphorylation. (B) Representative Western blot showing the impacts of inhibiting CAMKII (KN62, KN93) or PKA (H89) on Shank3 phosphorylation at baseline and upon TTX treatment. (C) Quantification of S1615 phosphorylation in (B) (two-way ANOVA with post-hoc Tukey’s test: DMSO vs. KN62, p>0.9999, DMSO vs. KN93, p=0.8148, DMSO vs. H89, p=0.9112, DMSO vs. picrotoxin (PTX), *p=0.0406, PTX vs. PTX/KN62, **p=0.0040, PTX vs. PTX/KN93, ****p<0.0001, PTX vs. PTX/H89, ****p<0.0001, n = 5 biological replicates). Dashed line indicates the DMSO control. (D) Quantification of PP2A activity after 1 hr TTX treatment (Un, n = 5, TTX, n = 5; paired t-test: **p=0.0018). (E) Quantification of PP2A activity after 24 hr TTX treatment (Un, n = 7, TTX, n = 7; paired t-test: *p=0.0129). (F, G) Western blot analyses showing changes in S1615 phosphorylation after 1 hr (F) or 24 hr (G) TTX treatment, with inhibition of PP2A by okadaic acid (OKA, 50 nM) during the

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Activity Assay, Phospho-proteomics, Western Blot, Control, Inhibition

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 5. PP2A activity is required for tetrodotoxin (TTX)-induced synaptic enrichment of Shank3. (A) Representative images of synaptic enrichment of endogenous Shank3 upon treatment with TTX and PP2A inhibitor fostriecin (FST) (scale bar = 10 µm). (B) Quantification of synaptic Shank3 intensity in (A) (number of neurons: DMSO, n = 26, FST, n = 28, TTX, n = 28, TTX/FST, n = 29; Kruskal–Wallis test with post-hoc Dunn’s tests: DMSO vs. FST, p>0.9999, DMSO vs. TTX, ***p=0.0002, FST vs. TTX/FST, p=0.1259, TTX vs. TTX/FST, p=0.1292). (C) Quantification of density of synapses containing Shank3 in (A) (Kruskal–Wallis test with post-hoc Dunn’s tests: DMSO vs. FST, p=0.9458, DMSO vs. TTX, **p=0.0051, FST vs. TTX/FST, p=0.2446, TTX vs. TTX/FST, *p=0.0273). (D) Representative images of synaptic enrichment of endogenous Shank3 upon treatment with TTX and PP1 inhibitor tautomycetin (TAUT) (scale bar = 10 µm). (E) Quantification of synaptic Shank3 intensity in (D) (number of neurons: DMSO, n = 26, TAUT, n = 21, TTX, n = 28, TTX/ TAUT, n = 32; Kruskal–Wallis test with post-hoc Dunn’s tests: DMSO vs. TAUT, *p=0.0315, DMSO vs. TTX, ***p=0.0006, TAUT vs. TTX/TAUT, ***p=0.0002, TTX vs. TTX/TAUT, *p=0.0392). (F) Quantification of density of synapses containing Shank3 in (D) (Kruskal–Wallis test with post-hoc Dunn’s tests: DMSO vs. TAUT, p=0.2450, DMSO vs. TTX, *p=0.0116, TAUT vs. TTX/TAUT, p=0.6552, TTX vs. TTX/TAUT, ***p=0.0007). Also see Figure 5—figure supplement 1 and Figure 5—source data 1.

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Activity Assay

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 6. Changes in the phosphorylation state of Shank3 are crucial for bidirectional synaptic scaling. (A, B) Representative miniature excitatory postsynaptic current (mEPSC) recordings from neurons overexpressing Shank3 WT (A) or DD mutant (B) during scaling up. (C) Quantification of average mEPSC amplitude in (A) (WT, n = 8, WT + tetrodotoxin [TTX], n = 9; unpaired two-tailed t-test: **p=0.0074). (D) Quantification of average mEPSC amplitude in (B) (number of neurons: DD, n = 12, DD + TTX, n = 14; unpaired two-tailed t-test: p=0.5708). (E, F) Representative traces of mEPSCs recorded from neurons overexpressing Shank3 WT (E) or AA mutant (F) during scaling down. (G) Quantification of average mEPSC amplitude in (E) (number of neurons: WT, n = 8, WT + bicuculline [BIC], n = 8; Mann–Whitney test: *p=0.0148). (H) Quantification of average mEPSC amplitude in (F) (AA, n = 9, AA + BIC, n = 14; unpaired two-tailed t-test: p=0.8612). Also see Figure 6—figure supplement 1, Figure 6—figure supplement 2, and Figure 6—source data 1.

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Phospho-proteomics, Mutagenesis, Two Tailed Test, MANN-WHITNEY

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 7. Brief PP2A inactivation reverses scaling up. (A) Representative images showing the effects of 1 hr fostriecin (FST) treatment on synaptic sGluA2 intensity in neurons expressing Shank3 WT or AA, after 24 hr of tetrodotoxin (TTX) to scale up synaptic strengths (scale bar = 10 µm). (B) Quantification of synaptic sGluA2 intensity in (A) (number of cells: WT/TTX, n = 22, WT/TTX/FST, n = 23, AA/TTX, n = 26, AA/TTX/FST, n = 25; Mann–Whitney test: WT/TTX vs. WT/TTX/FST, ***p=0.0007, AA/TTX vs. AA/TTX/FST, p=0.3739). (C) Quantification of synaptic Shank3 intensity in (A) (Mann–Whitney test: WT/TTX vs. WT/TTX/FST, **p=0.0090, AA/TTX vs. AA/TTX/FST, p=0.7296). (D) Quantification of the density of puncta containing sGluA2 and Shank3 (Mann–Whitney test: WT/TTX vs. WT/TTX/FST, **p=0.0016, AA/TTX vs. AA/TTX/FST, p=0.7017). Each data point indicates a cell, and the total number (n) was pooled from five independent experiments. Also see Figure 7—source data 1.

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Expressing, MANN-WHITNEY

Journal: Tissue Barriers

Article Title: GRP75 as a functional element of cholix transcytosis

doi: 10.1080/21688370.2022.2039003

Figure Lengend Snippet: GRP75 is an interactive partner of cholix. Polarized monolayers of Caco-2 cells were exposed apically to nontoxic full-length Chx (ntChx) anchored through a C-terminal hexa-histidine sequence to 100 nm diameter magnetic beads. After 15 min, cell membranes were isolated and separated into non-magnetic and magnetic fractions. a, 1-D SDS-PAGE gel of non-magnetic and magnetic captured membrane fractions. b, 2-D gel separation of proteins present in non-magnetic and magnetic capture membrane fractions with location of spot 16 identified by mass spectrometry as GRP75. c, Pull-down (PD) using ntChx probed with antibodies to either GRP75 or human growth hormone (hGH). d, Immunoprecipitation of Caco-2 cell lysate using ntChx probed with anti-GRP75 or control antibodies. e, 2-D Western blot of magnetic capture membrane fraction probed with anti-GRP75. f, ELISA-based, neutral pH, format where bound ntChx or the control protein bovine serum albumin (BSA) was used to capture GRP75, heat shock protein 60 (HSP60), glucose-regulated protein 78 (GRP78), or HSP90.

Article Snippet: Western blotting was performed using rabbit anti-human GRP75 as primary antibody followed by using Cy3-labeled Donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, 1;2500).

Techniques: Sequencing, Magnetic Beads, Isolation, SDS Page, Membrane, Mass Spectrometry, Immunoprecipitation, Control, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Tissue Barriers

Article Title: GRP75 as a functional element of cholix transcytosis

doi: 10.1080/21688370.2022.2039003

Figure Lengend Snippet: GRP75 and perlecan are functionally involved in cholix transcytosis. Expression of three proteins identified by ntChx-magnetic bead capture were suppressed using a CRISPR/cas 9-mediated knockdown protocol that targeted either GRP75, HSPG2 , or KRT8 in Caco-2 cells. Confluent monolayers of the resulting cell lines having decreased levels of a, GRP75, b, perlecan, or c, cytokeratin 8 (CT8) were evaluated for their capacity for Chx-mediated transcytosis using Chx266-hGH and for the nonspecific movement of the control protein hGH over a 60 min time period. d, Transcytosis of Chx266-hGH across confluent monolayers of parent Caco-2 cells was evaluated following a simultaneous apical application of a monoclonal antibody against GRP75 or perlecan, with an isotype antibody recognizing interleukin (IL)-10 serving as a control. Densitometry measurements of individual Western blot bands are shown for graphical comparison.

Article Snippet: Western blotting was performed using rabbit anti-human GRP75 as primary antibody followed by using Cy3-labeled Donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, 1;2500).

Techniques: Expressing, CRISPR, Knockdown, Control, Western Blot, Comparison

Journal: Tissue Barriers

Article Title: GRP75 as a functional element of cholix transcytosis

doi: 10.1080/21688370.2022.2039003

Figure Lengend Snippet: GRP75 associates with cholix in apical endosomes. Immunofluorescence microscopy was performed on rat jejunum over a time course following intraluminal injection (ILI) of AMT-101. Co-localization of the human interleukin (IL)-10 element of AMT-101 with TMEM132A (a, c) or GRP75 (b, d) at 1- or 5-min post ILI. Apical (luminal) epithelial membrane (open arrow); basal epithelial cell surface-basement membrane demarcation (dashed line); lamina propria ( l-p ); goblet cell (g). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue).

Article Snippet: Western blotting was performed using rabbit anti-human GRP75 as primary antibody followed by using Cy3-labeled Donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, 1;2500).

Techniques: Immunofluorescence, Microscopy, Injection, Membrane, Staining

Journal: Tissue Barriers

Article Title: GRP75 as a functional element of cholix transcytosis

doi: 10.1080/21688370.2022.2039003

Figure Lengend Snippet: Relationship of GRP75 to GRP78 and TMEM132A. Immunofluorescence microscopy was performed on rat jejunum over a time course following intraluminal injection (ILI) of Chx266-hGH. Co-localization events detecting the human growth hormone (hGH) element of Chx266-hGH with a, TMEM132A or b, GRP78 compared to the co-localization of c, TMEM132A and GRP78 at 5 min post ILI of Chx266-hGH. Apical (luminal) epithelial membrane (open arrow); basal epithelial cell surface-basement membrane demarcation (dashed line); lamina propria ( l-p ); goblet cell (g). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue).

Article Snippet: Western blotting was performed using rabbit anti-human GRP75 as primary antibody followed by using Cy3-labeled Donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, 1;2500).

Techniques: Immunofluorescence, Microscopy, Injection, Membrane, Staining

Journal: Tissue Barriers

Article Title: GRP75 as a functional element of cholix transcytosis

doi: 10.1080/21688370.2022.2039003

Figure Lengend Snippet: GRP75 does not associate with cholix in basal vesicular structures. Immunofluorescence microscopy was performed on rat jejunum after intraluminal injection (ILI) of Chx266-hGH. The human growth hormone (hGH) element of Chx266-hGH was co-localized with early endosomal antigen 1 (EEA1; a, c) or GRP75 (b, d) at 5- or 15-min post ILI. Apical (luminal) epithelial membrane (open arrow); basal epithelial cell surface-basement membrane demarcation (dashed line); lamina propria ( l-p ); goblet cell (g). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue).

Article Snippet: Western blotting was performed using rabbit anti-human GRP75 as primary antibody followed by using Cy3-labeled Donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, 1;2500).

Techniques: Immunofluorescence, Microscopy, Injection, Membrane, Staining

Journal: Tissue Barriers

Article Title: GRP75 as a functional element of cholix transcytosis

doi: 10.1080/21688370.2022.2039003

Figure Lengend Snippet: Cholix trafficking involves LMAN1-positive compartments. Immunofluorescence microscopy was performed on rat jejunum after intraluminal injection (ILI) of Chx266-hGH. The human growth hormone (hGH) element of Chx266-hGH was co-localized with a, early endosomal antigen 1 (EEA1) or b, LMAN1 at 5 min post ILI. c, Co-localization of EEA1 and LMAN1 at 5 min post ILI. Co-localization of hGH with d, GRP75 or e, LMAN1 at 15 min post ILI of Chx266-hGH. f, Co-localization of GRP75 and LMAN1 at 15 min post ILI of Chx266-hGH. Apical (luminal) epithelial membrane (open arrow); basal epithelial cell surface-basement membrane demarcation (dashed line); lamina propria ( l-p ); goblet cell (g). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue).

Article Snippet: Western blotting was performed using rabbit anti-human GRP75 as primary antibody followed by using Cy3-labeled Donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, 1;2500).

Techniques: Immunofluorescence, Microscopy, Injection, Membrane, Staining

Journal: Tissue Barriers

Article Title: GRP75 as a functional element of cholix transcytosis

doi: 10.1080/21688370.2022.2039003

Figure Lengend Snippet: GRP75 intersects with furin differently than with LMAN1. Immunofluorescence microscopy was performed on rat jejunum after intraluminal injection (ILI) of Chx266-hGH. The human growth hormone (hGH) element of Chx266-hGH was co-localized with a, furin or b, GRP75 at 15 min post ILI. c, Co-localization of furin and GRP75 at 15 min post ILI. Co-localization of hGH with d, furin or e, LMAN1 at 15 min post ILI of Chx266-hGH. f, Co-localization of furin and LMAN1 at 15 min post ILI of Chx266-hGH. Apical (luminal) epithelial membrane (open arrow); basal epithelial cell surface-basement membrane demarcation (dashed line); lamina propria ( l-p ); goblet cell (g). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue).

Article Snippet: Western blotting was performed using rabbit anti-human GRP75 as primary antibody followed by using Cy3-labeled Donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, 1;2500).

Techniques: Immunofluorescence, Microscopy, Injection, Membrane, Staining

Journal: Tissue Barriers

Article Title: GRP75 as a functional element of cholix transcytosis

doi: 10.1080/21688370.2022.2039003

Figure Lengend Snippet: Diagram of events associated with cholix apical to basal transcytosis with interaction partners. Highlighted steps are explained in the text with greater detail. (1) Cholix enters at the apical plasma membrane in the microvilli area where it interacts with TMEM132A and furin. (2) Cholix enters an early endosomal compartment where it traffics preferentially to a late endosome rather than a recycling pathway. (3) Instead of continuing along the lysosomal degradation pathway, cholix interacts with GRP75 and moves to a sorting endosome. (4) Reorganization of LMAN1 from the endoplasmic reticulum intermediate Golgi complex (ERGIC) to apical sorting vesicles. (5) In this location, LMAN1 can intersect with cholix delivered to this site with furin and GRP75. (6) LMAN1 and furin appear to return with cholix to a supranuclear region of the cell consistent with location of the ERGIC. (7) Reorganization of LMAN1 from the ERGIC to the basal vesicular compartment provides a route for cholix to this region of the cell. (8) Cholix in complex with LMAN1 enters sorting endosomes in the basal vesicular compartment. (9) Within basal sorting endosomes, cholix/LMAN1 intersects with perlecan in a recycling endosome, which delivers it to the basal plasma membrane, resulting in cholix exocytosis and completion of the apical to basal transcytosis process. (10) Perlecan recycles to the basal sorting endosome where it can engage cholix trafficked to this site with LMAN1. (11) Unlike in the apical vesicular compartment, GRP75 and furin present in the basal vesicular compartment are not associated with cholix in this region of enterocytes.

Article Snippet: Western blotting was performed using rabbit anti-human GRP75 as primary antibody followed by using Cy3-labeled Donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, 1;2500).

Techniques: Clinical Proteomics, Membrane