rabbit polyclonal anti myc tag proteintech  (Proteintech)

Journal: eLife

Article Title: Multiple ciliary localization signals control INPP5E ciliary targeting

doi: 10.7554/elife.78383

Figure Lengend Snippet: Figure 1. INPP5E catalytic domain encompasses FDRxLYL motif and is required for ciliary targeting. (a) Top diagram represents full length human INPP5E protein sequence (aa 1–644). Depicted are the proline-rich region (aa 10–242, Uniprot), the previously reported ciliary localization signal (FDRxLYL, aa 609–615 Humbert et al., 2012), and the CaaX box driving farnesylation (aa 641–644). Also shown is the inositol polyphosphate 5-phosphatase catalytic domain, whose most conserved core corresponds to InterPro domain IPR000300 (aa 297–599), but which actually spans aa

Article Snippet: DOI: https://doi.org/10.7554/eLife.78383 28 of 43 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- Myc (rabbit polyclonal) Proteintech Cat # 16286–1- AP WB: 1:1000 Antibody Anti- INPP5E (rabbit polyclonal) Proteintech Cat # 17797–1- AP IF: 1:100 Antibody Anti- γ-tubulin (goat polyclonal) Santa Cruz Cat # sc- 7396 (discontinued) IF: 1:200 Antibody Alexa Fluor 488 donkey anti- rabbit IgG (donkey polyclonal) Thermo Fisher Cat # A21206 IF: 1:10,000 Antibody Alexa Fluor 555 donkey anti- mouse IgG (donkey polyclonal) Thermo Fisher Cat # A31570 IF: 1:10,000 Antibody Alexa Fluor 647 donkey anti- goat IgG (donkey polyclonal) Thermo Fisher Cat # A21447 IF: 1:10,000 Antibody Alexa Fluor 488 goat anti- mouse IgG2a (goat polyclonal) Thermo Fisher Cat # A21131 IF: 1:10,000 Antibody Alexa Fluor 555 goat anti- mouse IgG1 (goat polyclonal) Thermo Fisher Cat # A21127 IF: 1:10,000 Antibody Alexa Fluor 555 goat anti- mouse IgG2b (goat polyclonal) Thermo Fisher Cat # A21147 IF: 1:10,000 Antibody Alexa Fluor 647 goat anti- mouse IgG2a (goat polyclonal) Thermo Fisher Cat # A21241 IF: 1:10,000 Antibody HRP- conjugated goat anti- mouse IgG (goat polyclonal) Thermo Fisher Cat # A16072 WB: 62 ng/ml Antibody HRP- conjugated goat anti- rabbit IgG (goat polyclonal) Thermo Fisher Cat # A16104 WB: 62 ng/ml Antibody GFP- Trap_MA magnetic agarose beads (alpaca monoclonal) Chromotek (Proteintech) Cat # gtma- 20 IP: 10 μl slurry for 500 μl lysate (1:50) Recombinant DNA reagent EGFP- INPP5E Jacoby et al., 2009 Human INPP5E NM_019892.6 (644 amino acids) XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (MORM) Jacoby et al., 2009 1–626 (MORM: Δ627–644) XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (D477N) Garcia- Gonzalo et al., 2015 D477N XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 623) This study 1–623 XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 621) This study 1–621 XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 618) This study 1–618 XhoI- KpnI cloning into pEGFP- C1 Continued Continued on next page Cilleros- Rodriguez, Martin- Morales et al. eLife 2022;11:e78383.

Techniques: Sequencing

Journal: eLife

Article Title: Multiple ciliary localization signals control INPP5E ciliary targeting

doi: 10.7554/elife.78383

Figure Lengend Snippet: Figure 2. W383 and FDRxLYL motifs act as CLSs on the catalytic domain surface. (a) Cilia localization of the indicated FDRxLYL mutants of EGFP-INPP5E was analyzed in hTERT-RPE1 cells as in Figure 1. Scale bars, 5 µm. (b) Magnification from INPP5E structure (PDB ID: 2xsw) showing the FDRxLYL motif residues (pink) and adjacent catalytic domain residues shown here to affect ciliary targeting (green). Distance between W383 and F609 is indicated in angstroms. Beta-sheets and alpha-helices are shown as yellow and orange ribbons, respectively. Notice active site region on top left (cyan). (c) Cilia localization of the indicated EGFP-INPP5E constructs was analyzed as in (a). In both cases, images are representative of at least two independent experiments per construct, with >30 transfected-cell cilia visualized per construct and experiment. Scale bars, 5 µm. (d) Percentage of positive cilia

Article Snippet: DOI: https://doi.org/10.7554/eLife.78383 28 of 43 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- Myc (rabbit polyclonal) Proteintech Cat # 16286–1- AP WB: 1:1000 Antibody Anti- INPP5E (rabbit polyclonal) Proteintech Cat # 17797–1- AP IF: 1:100 Antibody Anti- γ-tubulin (goat polyclonal) Santa Cruz Cat # sc- 7396 (discontinued) IF: 1:200 Antibody Alexa Fluor 488 donkey anti- rabbit IgG (donkey polyclonal) Thermo Fisher Cat # A21206 IF: 1:10,000 Antibody Alexa Fluor 555 donkey anti- mouse IgG (donkey polyclonal) Thermo Fisher Cat # A31570 IF: 1:10,000 Antibody Alexa Fluor 647 donkey anti- goat IgG (donkey polyclonal) Thermo Fisher Cat # A21447 IF: 1:10,000 Antibody Alexa Fluor 488 goat anti- mouse IgG2a (goat polyclonal) Thermo Fisher Cat # A21131 IF: 1:10,000 Antibody Alexa Fluor 555 goat anti- mouse IgG1 (goat polyclonal) Thermo Fisher Cat # A21127 IF: 1:10,000 Antibody Alexa Fluor 555 goat anti- mouse IgG2b (goat polyclonal) Thermo Fisher Cat # A21147 IF: 1:10,000 Antibody Alexa Fluor 647 goat anti- mouse IgG2a (goat polyclonal) Thermo Fisher Cat # A21241 IF: 1:10,000 Antibody HRP- conjugated goat anti- mouse IgG (goat polyclonal) Thermo Fisher Cat # A16072 WB: 62 ng/ml Antibody HRP- conjugated goat anti- rabbit IgG (goat polyclonal) Thermo Fisher Cat # A16104 WB: 62 ng/ml Antibody GFP- Trap_MA magnetic agarose beads (alpaca monoclonal) Chromotek (Proteintech) Cat # gtma- 20 IP: 10 μl slurry for 500 μl lysate (1:50) Recombinant DNA reagent EGFP- INPP5E Jacoby et al., 2009 Human INPP5E NM_019892.6 (644 amino acids) XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (MORM) Jacoby et al., 2009 1–626 (MORM: Δ627–644) XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (D477N) Garcia- Gonzalo et al., 2015 D477N XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 623) This study 1–623 XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 621) This study 1–621 XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 618) This study 1–618 XhoI- KpnI cloning into pEGFP- C1 Continued Continued on next page Cilleros- Rodriguez, Martin- Morales et al. eLife 2022;11:e78383.

Techniques: Construct, Transfection

Journal: eLife

Article Title: Multiple ciliary localization signals control INPP5E ciliary targeting

doi: 10.7554/elife.78383

Figure Lengend Snippet: Figure 3. The LLxPIR motif is a novel CLS that cooperates with the CaaX box to mediate INPP5E ciliary targeting. (a) Schema of full length human INPP5E and its mutants used in (b–c). Cilia localization of each mutant is indicated on the right. (b) Cilia localization of WT and indicated mutants was analyzed in hTERT-RPE1 cells as in Figures 1–2. Images are representative of n=3 independent experiments, with >30 transfected-cell cilia visualized per construct and experiment. Scale bar, 5 µm. (c) Quantitation of data from (b). The percentage of positive cilia in transfected cells is shown for the

Article Snippet: DOI: https://doi.org/10.7554/eLife.78383 28 of 43 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- Myc (rabbit polyclonal) Proteintech Cat # 16286–1- AP WB: 1:1000 Antibody Anti- INPP5E (rabbit polyclonal) Proteintech Cat # 17797–1- AP IF: 1:100 Antibody Anti- γ-tubulin (goat polyclonal) Santa Cruz Cat # sc- 7396 (discontinued) IF: 1:200 Antibody Alexa Fluor 488 donkey anti- rabbit IgG (donkey polyclonal) Thermo Fisher Cat # A21206 IF: 1:10,000 Antibody Alexa Fluor 555 donkey anti- mouse IgG (donkey polyclonal) Thermo Fisher Cat # A31570 IF: 1:10,000 Antibody Alexa Fluor 647 donkey anti- goat IgG (donkey polyclonal) Thermo Fisher Cat # A21447 IF: 1:10,000 Antibody Alexa Fluor 488 goat anti- mouse IgG2a (goat polyclonal) Thermo Fisher Cat # A21131 IF: 1:10,000 Antibody Alexa Fluor 555 goat anti- mouse IgG1 (goat polyclonal) Thermo Fisher Cat # A21127 IF: 1:10,000 Antibody Alexa Fluor 555 goat anti- mouse IgG2b (goat polyclonal) Thermo Fisher Cat # A21147 IF: 1:10,000 Antibody Alexa Fluor 647 goat anti- mouse IgG2a (goat polyclonal) Thermo Fisher Cat # A21241 IF: 1:10,000 Antibody HRP- conjugated goat anti- mouse IgG (goat polyclonal) Thermo Fisher Cat # A16072 WB: 62 ng/ml Antibody HRP- conjugated goat anti- rabbit IgG (goat polyclonal) Thermo Fisher Cat # A16104 WB: 62 ng/ml Antibody GFP- Trap_MA magnetic agarose beads (alpaca monoclonal) Chromotek (Proteintech) Cat # gtma- 20 IP: 10 μl slurry for 500 μl lysate (1:50) Recombinant DNA reagent EGFP- INPP5E Jacoby et al., 2009 Human INPP5E NM_019892.6 (644 amino acids) XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (MORM) Jacoby et al., 2009 1–626 (MORM: Δ627–644) XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (D477N) Garcia- Gonzalo et al., 2015 D477N XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 623) This study 1–623 XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 621) This study 1–621 XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 618) This study 1–618 XhoI- KpnI cloning into pEGFP- C1 Continued Continued on next page Cilleros- Rodriguez, Martin- Morales et al. eLife 2022;11:e78383.

Techniques: Mutagenesis, Transfection, Construct, Quantitation Assay

Journal: eLife

Article Title: Multiple ciliary localization signals control INPP5E ciliary targeting

doi: 10.7554/elife.78383

Figure Lengend Snippet: Figure 5. A subset of Joubert syndrome INPP5E mutations abolishes ciliary targeting. (a) Table of INPP5E ciliopathy mutations analyzed here. JBTS: Joubert syndrome; LCA: Leber congenital amaurosis; hom: homozygous; comp het: compound heterozygous. (b) Schema of INPP5E protein sequence indicating the locations of the ciliopathy mutations from (c) relative to its four CLSs, its catalytic domain (cyan) and its N-terminal proline-rich region (green). (c) Ciliary localization of mutants from (a–b) was analyzed in hTERT-RPE1 cells as in Figures 1–3. Images are representative of at least two

Article Snippet: DOI: https://doi.org/10.7554/eLife.78383 28 of 43 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- Myc (rabbit polyclonal) Proteintech Cat # 16286–1- AP WB: 1:1000 Antibody Anti- INPP5E (rabbit polyclonal) Proteintech Cat # 17797–1- AP IF: 1:100 Antibody Anti- γ-tubulin (goat polyclonal) Santa Cruz Cat # sc- 7396 (discontinued) IF: 1:200 Antibody Alexa Fluor 488 donkey anti- rabbit IgG (donkey polyclonal) Thermo Fisher Cat # A21206 IF: 1:10,000 Antibody Alexa Fluor 555 donkey anti- mouse IgG (donkey polyclonal) Thermo Fisher Cat # A31570 IF: 1:10,000 Antibody Alexa Fluor 647 donkey anti- goat IgG (donkey polyclonal) Thermo Fisher Cat # A21447 IF: 1:10,000 Antibody Alexa Fluor 488 goat anti- mouse IgG2a (goat polyclonal) Thermo Fisher Cat # A21131 IF: 1:10,000 Antibody Alexa Fluor 555 goat anti- mouse IgG1 (goat polyclonal) Thermo Fisher Cat # A21127 IF: 1:10,000 Antibody Alexa Fluor 555 goat anti- mouse IgG2b (goat polyclonal) Thermo Fisher Cat # A21147 IF: 1:10,000 Antibody Alexa Fluor 647 goat anti- mouse IgG2a (goat polyclonal) Thermo Fisher Cat # A21241 IF: 1:10,000 Antibody HRP- conjugated goat anti- mouse IgG (goat polyclonal) Thermo Fisher Cat # A16072 WB: 62 ng/ml Antibody HRP- conjugated goat anti- rabbit IgG (goat polyclonal) Thermo Fisher Cat # A16104 WB: 62 ng/ml Antibody GFP- Trap_MA magnetic agarose beads (alpaca monoclonal) Chromotek (Proteintech) Cat # gtma- 20 IP: 10 μl slurry for 500 μl lysate (1:50) Recombinant DNA reagent EGFP- INPP5E Jacoby et al., 2009 Human INPP5E NM_019892.6 (644 amino acids) XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (MORM) Jacoby et al., 2009 1–626 (MORM: Δ627–644) XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (D477N) Garcia- Gonzalo et al., 2015 D477N XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 623) This study 1–623 XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 621) This study 1–621 XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 618) This study 1–618 XhoI- KpnI cloning into pEGFP- C1 Continued Continued on next page Cilleros- Rodriguez, Martin- Morales et al. eLife 2022;11:e78383.

Techniques: Sequencing

Journal: eLife

Article Title: Multiple ciliary localization signals control INPP5E ciliary targeting

doi: 10.7554/elife.78383

Figure Lengend Snippet: Figure 6. CLS4 promotes INPP5E binding to PDE6D, RPGR and ARL13B. (a) The indicated EGFP-INPP5E variants were coexpressed in HEK293T cells with Flag-PDE6D, as indicated. Lysates were immunoprecipitated with GFP-Trap beads and analyzed by Western blot with the indicated antibodies. Molecular weight markers in kilodaltons are shown on the right. Quantitation of Flag-PDE6D co-immunoprecipitation (co-IP), as percentage relative to WT, is shown at the bottom. Quantitations are normalized relative to both immunoprecipitated EGFP constructs and lysate amounts of Flag-PDE6D. (b)

Article Snippet: DOI: https://doi.org/10.7554/eLife.78383 28 of 43 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- Myc (rabbit polyclonal) Proteintech Cat # 16286–1- AP WB: 1:1000 Antibody Anti- INPP5E (rabbit polyclonal) Proteintech Cat # 17797–1- AP IF: 1:100 Antibody Anti- γ-tubulin (goat polyclonal) Santa Cruz Cat # sc- 7396 (discontinued) IF: 1:200 Antibody Alexa Fluor 488 donkey anti- rabbit IgG (donkey polyclonal) Thermo Fisher Cat # A21206 IF: 1:10,000 Antibody Alexa Fluor 555 donkey anti- mouse IgG (donkey polyclonal) Thermo Fisher Cat # A31570 IF: 1:10,000 Antibody Alexa Fluor 647 donkey anti- goat IgG (donkey polyclonal) Thermo Fisher Cat # A21447 IF: 1:10,000 Antibody Alexa Fluor 488 goat anti- mouse IgG2a (goat polyclonal) Thermo Fisher Cat # A21131 IF: 1:10,000 Antibody Alexa Fluor 555 goat anti- mouse IgG1 (goat polyclonal) Thermo Fisher Cat # A21127 IF: 1:10,000 Antibody Alexa Fluor 555 goat anti- mouse IgG2b (goat polyclonal) Thermo Fisher Cat # A21147 IF: 1:10,000 Antibody Alexa Fluor 647 goat anti- mouse IgG2a (goat polyclonal) Thermo Fisher Cat # A21241 IF: 1:10,000 Antibody HRP- conjugated goat anti- mouse IgG (goat polyclonal) Thermo Fisher Cat # A16072 WB: 62 ng/ml Antibody HRP- conjugated goat anti- rabbit IgG (goat polyclonal) Thermo Fisher Cat # A16104 WB: 62 ng/ml Antibody GFP- Trap_MA magnetic agarose beads (alpaca monoclonal) Chromotek (Proteintech) Cat # gtma- 20 IP: 10 μl slurry for 500 μl lysate (1:50) Recombinant DNA reagent EGFP- INPP5E Jacoby et al., 2009 Human INPP5E NM_019892.6 (644 amino acids) XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (MORM) Jacoby et al., 2009 1–626 (MORM: Δ627–644) XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (D477N) Garcia- Gonzalo et al., 2015 D477N XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 623) This study 1–623 XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 621) This study 1–621 XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 618) This study 1–618 XhoI- KpnI cloning into pEGFP- C1 Continued Continued on next page Cilleros- Rodriguez, Martin- Morales et al. eLife 2022;11:e78383.

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Molecular Weight, Quantitation Assay, Co-Immunoprecipitation Assay, Construct

Journal: eLife

Article Title: Multiple ciliary localization signals control INPP5E ciliary targeting

doi: 10.7554/elife.78383

Figure Lengend Snippet: Figure 7. CLS2 and CLS3 regulate INPP5E binding to TULP3 and CEP164. (a) The indicated EGFP-INPP5E variants were coexpressed in HEK293T cells with TULP3-myc as indicated. Lysates were immunoprecipitated with GFP-Trap beads and analyzed by Western blot with TULP3 and EGFP antibodies, as indicated. (b) Quantitation of TULP3-myc co-IP by the indicated EGFP-INPP5E constructs in HEK293T cells. Co-IP levels, expressed as percentage of WT, are normalized by the amounts of both immunoprecipitated EGFP-INPP5Es and TULP3-myc lysate levels. Data are mean ± SEM of n=7,7,4,7,7,4,4 independent experiments and were analyzed by one-way ANOVA followed by Tukey multiple comparisons tests. Significance relative to WT is shown

Article Snippet: DOI: https://doi.org/10.7554/eLife.78383 28 of 43 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- Myc (rabbit polyclonal) Proteintech Cat # 16286–1- AP WB: 1:1000 Antibody Anti- INPP5E (rabbit polyclonal) Proteintech Cat # 17797–1- AP IF: 1:100 Antibody Anti- γ-tubulin (goat polyclonal) Santa Cruz Cat # sc- 7396 (discontinued) IF: 1:200 Antibody Alexa Fluor 488 donkey anti- rabbit IgG (donkey polyclonal) Thermo Fisher Cat # A21206 IF: 1:10,000 Antibody Alexa Fluor 555 donkey anti- mouse IgG (donkey polyclonal) Thermo Fisher Cat # A31570 IF: 1:10,000 Antibody Alexa Fluor 647 donkey anti- goat IgG (donkey polyclonal) Thermo Fisher Cat # A21447 IF: 1:10,000 Antibody Alexa Fluor 488 goat anti- mouse IgG2a (goat polyclonal) Thermo Fisher Cat # A21131 IF: 1:10,000 Antibody Alexa Fluor 555 goat anti- mouse IgG1 (goat polyclonal) Thermo Fisher Cat # A21127 IF: 1:10,000 Antibody Alexa Fluor 555 goat anti- mouse IgG2b (goat polyclonal) Thermo Fisher Cat # A21147 IF: 1:10,000 Antibody Alexa Fluor 647 goat anti- mouse IgG2a (goat polyclonal) Thermo Fisher Cat # A21241 IF: 1:10,000 Antibody HRP- conjugated goat anti- mouse IgG (goat polyclonal) Thermo Fisher Cat # A16072 WB: 62 ng/ml Antibody HRP- conjugated goat anti- rabbit IgG (goat polyclonal) Thermo Fisher Cat # A16104 WB: 62 ng/ml Antibody GFP- Trap_MA magnetic agarose beads (alpaca monoclonal) Chromotek (Proteintech) Cat # gtma- 20 IP: 10 μl slurry for 500 μl lysate (1:50) Recombinant DNA reagent EGFP- INPP5E Jacoby et al., 2009 Human INPP5E NM_019892.6 (644 amino acids) XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (MORM) Jacoby et al., 2009 1–626 (MORM: Δ627–644) XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (D477N) Garcia- Gonzalo et al., 2015 D477N XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 623) This study 1–623 XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 621) This study 1–621 XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 618) This study 1–618 XhoI- KpnI cloning into pEGFP- C1 Continued Continued on next page Cilleros- Rodriguez, Martin- Morales et al. eLife 2022;11:e78383.

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Quantitation Assay, Co-Immunoprecipitation Assay, Construct

Journal: eLife

Article Title: Multiple ciliary localization signals control INPP5E ciliary targeting

doi: 10.7554/elife.78383

Figure Lengend Snippet: Figure 8. CLS1 and CLS4 jointly modulate the ATG16L1-INPP5E interaction. (a) The indicated EGFP-INPP5E constructs were coexpressed in HEK293T cells with Flag-ATG16L1, as shown. Lysates were immunoprecipitated with GFP-Trap beads and analyzed by Western blot with the indicated antibodies. (b) Quantitation of Flag-ATG16L1 co-IP by the indicated EGFP-INPP5E constructs in HEK293T cells. Co-IP levels, expressed as percentage of WT, are normalized by the amounts of both immunoprecipitated EGFP-INPP5Es and Flag-ATG16L1 lysate levels. Data are mean ± SEM of n=3 independent experiments and were analyzed by one-way ANOVA followed by Dunnett tests relative to WT. Significance is shown as p<0.01(**). (c) Schema of INPP5E structure depicting CLS1-4 and the proteins through which they regulate INPP5E ciliary targeting, as shown herein.

Article Snippet: DOI: https://doi.org/10.7554/eLife.78383 28 of 43 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- Myc (rabbit polyclonal) Proteintech Cat # 16286–1- AP WB: 1:1000 Antibody Anti- INPP5E (rabbit polyclonal) Proteintech Cat # 17797–1- AP IF: 1:100 Antibody Anti- γ-tubulin (goat polyclonal) Santa Cruz Cat # sc- 7396 (discontinued) IF: 1:200 Antibody Alexa Fluor 488 donkey anti- rabbit IgG (donkey polyclonal) Thermo Fisher Cat # A21206 IF: 1:10,000 Antibody Alexa Fluor 555 donkey anti- mouse IgG (donkey polyclonal) Thermo Fisher Cat # A31570 IF: 1:10,000 Antibody Alexa Fluor 647 donkey anti- goat IgG (donkey polyclonal) Thermo Fisher Cat # A21447 IF: 1:10,000 Antibody Alexa Fluor 488 goat anti- mouse IgG2a (goat polyclonal) Thermo Fisher Cat # A21131 IF: 1:10,000 Antibody Alexa Fluor 555 goat anti- mouse IgG1 (goat polyclonal) Thermo Fisher Cat # A21127 IF: 1:10,000 Antibody Alexa Fluor 555 goat anti- mouse IgG2b (goat polyclonal) Thermo Fisher Cat # A21147 IF: 1:10,000 Antibody Alexa Fluor 647 goat anti- mouse IgG2a (goat polyclonal) Thermo Fisher Cat # A21241 IF: 1:10,000 Antibody HRP- conjugated goat anti- mouse IgG (goat polyclonal) Thermo Fisher Cat # A16072 WB: 62 ng/ml Antibody HRP- conjugated goat anti- rabbit IgG (goat polyclonal) Thermo Fisher Cat # A16104 WB: 62 ng/ml Antibody GFP- Trap_MA magnetic agarose beads (alpaca monoclonal) Chromotek (Proteintech) Cat # gtma- 20 IP: 10 μl slurry for 500 μl lysate (1:50) Recombinant DNA reagent EGFP- INPP5E Jacoby et al., 2009 Human INPP5E NM_019892.6 (644 amino acids) XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (MORM) Jacoby et al., 2009 1–626 (MORM: Δ627–644) XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (D477N) Garcia- Gonzalo et al., 2015 D477N XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 623) This study 1–623 XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 621) This study 1–621 XhoI- KpnI cloning into pEGFP- C1 Recombinant DNA reagent EGFP- INPP5E (1- 618) This study 1–618 XhoI- KpnI cloning into pEGFP- C1 Continued Continued on next page Cilleros- Rodriguez, Martin- Morales et al. eLife 2022;11:e78383.

Techniques: Construct, Immunoprecipitation, Western Blot, Quantitation Assay, Co-Immunoprecipitation Assay