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rabbit polyclonal antibodies against fip200 (Proteintech)

Name: rabbit polyclonal antibodies against fip200
Brand: Proteintech
Rating: 96 (148 reviews)

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Proteintech rabbit polyclonal antibodies against fip200

( A ) Structure of the <t>ULK1–FIP200</t> moiety of the ULK1–ATG13–FIP200 core complex in . The right panel represents the surface model of FIP200 with coloring based on the electrostatic potentials (blue and red indicate positive and negative potentials, respectively). Dotted squares indicate the regions displayed in ( B ). ( B ) Close-up view of the interactions between ULK1 MIT1 and FIP200 (top) and between ULK1 MIT2 and FIP200 (bottom). Left and right indicate AlphaFold2 and cryo-EM (PDB 8SOI) models. ( C ) In vitro pull-down assay between GST-ULK1 (636–1050 aa) WT or FIP2A mutant with MBP-FIP200 (1–634 aa). ( D ) Relative amounts of precipitated MBP-FIP200 in ( C ) were calculated. Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed using Tukey’s multiple comparisons test. ( E ) Effect of the ULK1 FIP2A mutation on the FIP200 interaction in vivo. Ulk1,2 DKO mouse embryonic fibroblasts (MEFs) stably expressing FLAG-tagged ULK1 WT or FIP2A mutant were immunoprecipitated with an anti-FLAG antibody and detected with anti-FIP200, anti-ATG13, and anti-FLAG antibodies. ( F ) Relative amounts of precipitated FIP200 (left) and ATG13 (right) in ( E ) were calculated. Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed using Tukey’s multiple comparisons test. ( G ) Halo-LC3 processing assay of ULK1 FIP2A-expressing cells. Ulk1,2 DKO MEFs stably expressing Halo-LC3 and FLAG-tagged ULK1 WT or FIP2A mutant were labeled for 15 min with 100 nm tetramethylrhodamine (TMR)-conjugated Halo ligand and incubated in starvation medium for 1 hr. Cell lysates were subjected to in-gel fluorescence detection. ( H ) Halo processing rate in ( G ). The band intensity of processed Halo and Halo-LC3 in each cell line was quantified, and the relative cleavage rate was calculated as FLAG-ULK1 WT-expressing cells as 1. Solid bars indicate the means, and dots indicate the data from three independent experiments. Data were statistically analyzed using Tukey’s multiple comparisons test. ( I ) Colocalization of FLAG-ULK1 WT or FIP2A mutant with FIP200. Ulk1,2 DKO MEFs stably expressing FLAG-tagged ULK1 WT or FIP2A mutant were immunostained with anti-FLAG and anti-FIP200 antibodies. Scale bar, 10 μm. Figure 3—source data 1. PDF file containing original western blots or SDS–PAGE for . Figure 3—source data 2. Original files for western blot or SDS–PAGE analysis displayed in . Figure 3—source data 3. Values used for preparation of the graph in .

Rabbit Polyclonal Antibodies Against Fip200, supplied by Proteintech, used in various techniques. Bioz Stars score: 96/100, based on 148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 96 stars, based on 148 article reviews

rabbit polyclonal antibodies against fip200 - by Bioz Stars, 2026-02

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1) Product Images from "The triad interaction of ULK1, ATG13, and FIP200 is required for ULK complex formation and autophagy"

Article Title: The triad interaction of ULK1, ATG13, and FIP200 is required for ULK complex formation and autophagy

Journal: eLife

doi: 10.7554/eLife.101531

( A ) Structure of the ULK1–FIP200 moiety of the ULK1–ATG13–FIP200 core complex in . The right panel represents the surface model of FIP200 with coloring based on the electrostatic potentials (blue and red indicate positive and negative potentials, respectively). Dotted squares indicate the regions displayed in ( B ). ( B ) Close-up view of the interactions between ULK1 MIT1 and FIP200 (top) and between ULK1 MIT2 and FIP200 (bottom). Left and right indicate AlphaFold2 and cryo-EM (PDB 8SOI) models. ( C ) In vitro pull-down assay between GST-ULK1 (636–1050 aa) WT or FIP2A mutant with MBP-FIP200 (1–634 aa). ( D ) Relative amounts of precipitated MBP-FIP200 in ( C ) were calculated. Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed using Tukey’s multiple comparisons test. ( E ) Effect of the ULK1 FIP2A mutation on the FIP200 interaction in vivo. Ulk1,2 DKO mouse embryonic fibroblasts (MEFs) stably expressing FLAG-tagged ULK1 WT or FIP2A mutant were immunoprecipitated with an anti-FLAG antibody and detected with anti-FIP200, anti-ATG13, and anti-FLAG antibodies. ( F ) Relative amounts of precipitated FIP200 (left) and ATG13 (right) in ( E ) were calculated. Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed using Tukey’s multiple comparisons test. ( G ) Halo-LC3 processing assay of ULK1 FIP2A-expressing cells. Ulk1,2 DKO MEFs stably expressing Halo-LC3 and FLAG-tagged ULK1 WT or FIP2A mutant were labeled for 15 min with 100 nm tetramethylrhodamine (TMR)-conjugated Halo ligand and incubated in starvation medium for 1 hr. Cell lysates were subjected to in-gel fluorescence detection. ( H ) Halo processing rate in ( G ). The band intensity of processed Halo and Halo-LC3 in each cell line was quantified, and the relative cleavage rate was calculated as FLAG-ULK1 WT-expressing cells as 1. Solid bars indicate the means, and dots indicate the data from three independent experiments. Data were statistically analyzed using Tukey’s multiple comparisons test. ( I ) Colocalization of FLAG-ULK1 WT or FIP2A mutant with FIP200. Ulk1,2 DKO MEFs stably expressing FLAG-tagged ULK1 WT or FIP2A mutant were immunostained with anti-FLAG and anti-FIP200 antibodies. Scale bar, 10 μm. Figure 3—source data 1. PDF file containing original western blots or SDS–PAGE for . Figure 3—source data 2. Original files for western blot or SDS–PAGE analysis displayed in . Figure 3—source data 3. Values used for preparation of the graph in .

Figure Legend Snippet: ( A ) Structure of the ULK1–FIP200 moiety of the ULK1–ATG13–FIP200 core complex in . The right panel represents the surface model of FIP200 with coloring based on the electrostatic potentials (blue and red indicate positive and negative potentials, respectively). Dotted squares indicate the regions displayed in ( B ). ( B ) Close-up view of the interactions between ULK1 MIT1 and FIP200 (top) and between ULK1 MIT2 and FIP200 (bottom). Left and right indicate AlphaFold2 and cryo-EM (PDB 8SOI) models. ( C ) In vitro pull-down assay between GST-ULK1 (636–1050 aa) WT or FIP2A mutant with MBP-FIP200 (1–634 aa). ( D ) Relative amounts of precipitated MBP-FIP200 in ( C ) were calculated. Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed using Tukey’s multiple comparisons test. ( E ) Effect of the ULK1 FIP2A mutation on the FIP200 interaction in vivo. Ulk1,2 DKO mouse embryonic fibroblasts (MEFs) stably expressing FLAG-tagged ULK1 WT or FIP2A mutant were immunoprecipitated with an anti-FLAG antibody and detected with anti-FIP200, anti-ATG13, and anti-FLAG antibodies. ( F ) Relative amounts of precipitated FIP200 (left) and ATG13 (right) in ( E ) were calculated. Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed using Tukey’s multiple comparisons test. ( G ) Halo-LC3 processing assay of ULK1 FIP2A-expressing cells. Ulk1,2 DKO MEFs stably expressing Halo-LC3 and FLAG-tagged ULK1 WT or FIP2A mutant were labeled for 15 min with 100 nm tetramethylrhodamine (TMR)-conjugated Halo ligand and incubated in starvation medium for 1 hr. Cell lysates were subjected to in-gel fluorescence detection. ( H ) Halo processing rate in ( G ). The band intensity of processed Halo and Halo-LC3 in each cell line was quantified, and the relative cleavage rate was calculated as FLAG-ULK1 WT-expressing cells as 1. Solid bars indicate the means, and dots indicate the data from three independent experiments. Data were statistically analyzed using Tukey’s multiple comparisons test. ( I ) Colocalization of FLAG-ULK1 WT or FIP2A mutant with FIP200. Ulk1,2 DKO MEFs stably expressing FLAG-tagged ULK1 WT or FIP2A mutant were immunostained with anti-FLAG and anti-FIP200 antibodies. Scale bar, 10 μm. Figure 3—source data 1. PDF file containing original western blots or SDS–PAGE for . Figure 3—source data 2. Original files for western blot or SDS–PAGE analysis displayed in . Figure 3—source data 3. Values used for preparation of the graph in .

Techniques Used: Cryo-EM Sample Prep, In Vitro, Pull Down Assay, Mutagenesis, In Vivo, Stable Transfection, Expressing, Immunoprecipitation, Labeling, Incubation, Fluorescence, Western Blot, SDS Page

( A ) AlphaFold2 model of the ULK1–ATG13 moiety of the ULK1–ATG13–FIP200 core complex in (left), Cryo-EM structure of the ULK1–ATG13 moiety of the ULK1–ATG13–FIP200 core complex (PDB 8SOI), and crystal structure of the yeast Atg1–Atg13 complex (right, PDB 4P1N). ( B ) Close-up view of the interactions between ATG13 MIM(N) and ULK1 MIT2 and between ATG13 MIM(C) and ULK1 MIT1 (right). ( C ) Isothermal titration calorimetry (ITC) results obtained by titration of MBP-ULK1 (636–1050 aa) into a solution of WT or ULK2A mutant of MBP-ATG13 (363–517 aa). Due to weak binding, the K D value for the ULK2A mutant was not accurately determined. ( D ) Effect of the ATG13–FIP3A mutation on endogenous ULK1 levels in vivo. WT or ATG13 KO HeLa cells stably expressing FLAG-tagged ATG13 WT or ULK2A mutant were lysed, and indicated proteins were detected by immunoblotting using anti-FIP200, anti-ULK1, and anti-FLAG antibodies. ( E ) Relative amounts of ULK1 in ( D ) were normalized with β-actin and calculated. Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed using Tukey’s multiple comparisons test. Figure 2—source data 1. PDF file containing original western blots for . Figure 2—source data 2. Original files for western blot analysis displayed in . Figure 2—source data 3. Values used for preparation of the graph in .

Figure Legend Snippet: ( A ) AlphaFold2 model of the ULK1–ATG13 moiety of the ULK1–ATG13–FIP200 core complex in (left), Cryo-EM structure of the ULK1–ATG13 moiety of the ULK1–ATG13–FIP200 core complex (PDB 8SOI), and crystal structure of the yeast Atg1–Atg13 complex (right, PDB 4P1N). ( B ) Close-up view of the interactions between ATG13 MIM(N) and ULK1 MIT2 and between ATG13 MIM(C) and ULK1 MIT1 (right). ( C ) Isothermal titration calorimetry (ITC) results obtained by titration of MBP-ULK1 (636–1050 aa) into a solution of WT or ULK2A mutant of MBP-ATG13 (363–517 aa). Due to weak binding, the K D value for the ULK2A mutant was not accurately determined. ( D ) Effect of the ATG13–FIP3A mutation on endogenous ULK1 levels in vivo. WT or ATG13 KO HeLa cells stably expressing FLAG-tagged ATG13 WT or ULK2A mutant were lysed, and indicated proteins were detected by immunoblotting using anti-FIP200, anti-ULK1, and anti-FLAG antibodies. ( E ) Relative amounts of ULK1 in ( D ) were normalized with β-actin and calculated. Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed using Tukey’s multiple comparisons test. Figure 2—source data 1. PDF file containing original western blots for . Figure 2—source data 2. Original files for western blot analysis displayed in . Figure 2—source data 3. Values used for preparation of the graph in .

Techniques Used: Cryo-EM Sample Prep, Isothermal Titration Calorimetry, Titration, Mutagenesis, Binding Assay, In Vivo, Stable Transfection, Expressing, Western Blot

( A ) Domain architecture of ULK1, ATG13, and FIP200. Regions used for the AlphaFold2 complex prediction are underlined. ( B ) Structure of the ULK1–ATG13–FIP200 core complex predicted by AlphaFold2. Flexible loop regions in FIP200 were removed from the figure for clarity. N and C indicate N- and C-terminal regions, respectively. ( C ) Close-up view of the interactions between ATG13 and FIP200. The bottom panels represent the surface model of FIP200 with the coloring based on the electrostatic potentials (blue and red indicate positive and negative potentials, respectively). ( D ) Isothermal titration calorimetry (ITC) results obtained by titration of MBP-ATG13 (363–517 aa) WT or FIP3A mutant into an FIP200 (1–634 aa) solution. ( E ) Effect of the ATG13 FIP3A mutation on the FIP200 interaction in vivo. ATG13 KO HeLa cells stably expressing FLAG-tagged ATG13 WT or FIP3A were immunoprecipitated with an anti-FLAG antibody and detected with anti-FIP200, anti-ULK1, and anti-FLAG antibodies. ( F ) Relative amounts of precipitated FIP200 in ( E ) were calculated. Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed using Tukey’s multiple comparisons test. Figure 1—source data 1. PDF file containing original western blots for . Figure 1—source data 2. Original files for western blot analysis displayed in . Figure 1—source data 3. Values used for preparation of the graph in .

Figure Legend Snippet: ( A ) Domain architecture of ULK1, ATG13, and FIP200. Regions used for the AlphaFold2 complex prediction are underlined. ( B ) Structure of the ULK1–ATG13–FIP200 core complex predicted by AlphaFold2. Flexible loop regions in FIP200 were removed from the figure for clarity. N and C indicate N- and C-terminal regions, respectively. ( C ) Close-up view of the interactions between ATG13 and FIP200. The bottom panels represent the surface model of FIP200 with the coloring based on the electrostatic potentials (blue and red indicate positive and negative potentials, respectively). ( D ) Isothermal titration calorimetry (ITC) results obtained by titration of MBP-ATG13 (363–517 aa) WT or FIP3A mutant into an FIP200 (1–634 aa) solution. ( E ) Effect of the ATG13 FIP3A mutation on the FIP200 interaction in vivo. ATG13 KO HeLa cells stably expressing FLAG-tagged ATG13 WT or FIP3A were immunoprecipitated with an anti-FLAG antibody and detected with anti-FIP200, anti-ULK1, and anti-FLAG antibodies. ( F ) Relative amounts of precipitated FIP200 in ( E ) were calculated. Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed using Tukey’s multiple comparisons test. Figure 1—source data 1. PDF file containing original western blots for . Figure 1—source data 2. Original files for western blot analysis displayed in . Figure 1—source data 3. Values used for preparation of the graph in .

Techniques Used: Isothermal Titration Calorimetry, Titration, Mutagenesis, In Vivo, Stable Transfection, Expressing, Immunoprecipitation, Western Blot

( A ) Comparison of ATG13 expression level. WT, ATG13 KO stably expressing ATG13-FLAG, and ATG13-FLAG KI HeLa cells were lysed, and indicated proteins were detected by immunoblotting using anti-ATG13, anti-FIP200, anti-ULK1, and anti-β-actin antibodies. ( B ) Relative amounts of ATG13 in ( A ) were calculated as WT HeLa cells as 1. Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed using Tukey’s multiple comparisons test. ( C ) Colocalization of ATG9A and FIP200 in ATG13-FLAG KI cells. Indicated KI cell lines expressing ATG9A-HA were cultured in the starvation medium for 1 hr and immunostained with anti-FLAG, anti-FIP200, and anti-p62 antibodies. Scale bar, 10 μm. ( D ) ULK1-dependent phosphorylation of ATG14 in ATG13-FLAG KI cell lines. WT, ATG13 KO and KI HeLa cell lines cultured in the starvation medium for 1 hr. Indicated proteins were detected by immunoblotting using anti-ATG14 phospho-S29, anti-ATG14, and anti-β-actin antibodies. ( E ) ATG14 phosphorylation rate in ( D ). The band intensity of p-ATG14 and ATG14 in each cell line was quantified, and the phosphorylation rate was calculated as WT HeLa cells as 1. Solid bars indicate the means, and dots indicate the data from three independent experiments. Data were statistically analyzed using Tukey’ s multiple comparisons test. Figure 4—figure supplement 1—source data 1. PDF file containing original western blots for . Figure 4—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 4—figure supplement 1—source data 3. Values used for preparation of the graph in .

Figure Legend Snippet: ( A ) Comparison of ATG13 expression level. WT, ATG13 KO stably expressing ATG13-FLAG, and ATG13-FLAG KI HeLa cells were lysed, and indicated proteins were detected by immunoblotting using anti-ATG13, anti-FIP200, anti-ULK1, and anti-β-actin antibodies. ( B ) Relative amounts of ATG13 in ( A ) were calculated as WT HeLa cells as 1. Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed using Tukey’s multiple comparisons test. ( C ) Colocalization of ATG9A and FIP200 in ATG13-FLAG KI cells. Indicated KI cell lines expressing ATG9A-HA were cultured in the starvation medium for 1 hr and immunostained with anti-FLAG, anti-FIP200, and anti-p62 antibodies. Scale bar, 10 μm. ( D ) ULK1-dependent phosphorylation of ATG14 in ATG13-FLAG KI cell lines. WT, ATG13 KO and KI HeLa cell lines cultured in the starvation medium for 1 hr. Indicated proteins were detected by immunoblotting using anti-ATG14 phospho-S29, anti-ATG14, and anti-β-actin antibodies. ( E ) ATG14 phosphorylation rate in ( D ). The band intensity of p-ATG14 and ATG14 in each cell line was quantified, and the phosphorylation rate was calculated as WT HeLa cells as 1. Solid bars indicate the means, and dots indicate the data from three independent experiments. Data were statistically analyzed using Tukey’ s multiple comparisons test. Figure 4—figure supplement 1—source data 1. PDF file containing original western blots for . Figure 4—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 4—figure supplement 1—source data 3. Values used for preparation of the graph in .

Techniques Used: Comparison, Expressing, Stable Transfection, Western Blot, Cell Culture, Phospho-proteomics

( A ) Schematic representation of the CRISPR–Cas9-mediated KI strategy of ATG13 mutations with FLAG tag. The C-terminally FLAG-tagged coding sequence after exon 14 of ATG13 with or without FIP3A, ULK2A, or FU5A mutations were knocked in exon 14 of the Homo sapiens ATG13 locus. As the KI cassette expresses NeoR under the hPGK1 promoter, clones that were successfully knocked in were selected by G418. Cas9-gRNA-targeted sites in the exon 14 of H. sapiens ATG13 locus are displayed in dark blue. The homology arm for KI is presented in magenta, and the ATG13 CDS and mutations in red and cyan, respectively. NeoR is displayed in brown. Scale bar, 0.5 kilobase pair (kb). ( B ) Immunoblot of ATG13-FLAG KI cell lines. WT, ATG13 KO, and indicated KI HeLa cells were lysed, and indicated proteins were detected by immunoblotting using anti-FIP200, anti-ULK1, and anti-FLAG antibodies. ( C ) Colocalization of endogenous levels of ATG13-FLAG mutants with FIP200. Indicated KI cell lines were cultured in the starvation medium for 1 hr and immunostained with anti-FLAG and anti-FIP200 antibodies. Scale bar, 10 μm. ( D ) Halo-LC3 processing assay of ATG13-FLAG KI cell lines. WT, ATG13 KO and KI HeLa cell lines were labeled for 15 min with 100 nm tetramethylrhodamine (TMR)-conjugated Halo ligand and incubated in starvation medium for 1 hr. Cell lysates were subjected to in-gel fluorescence detection. ( E ) Halo processing rate in ( D ). The band intensity of processed Halo and Halo-LC3 in each cell line was quantified, and the relative cleavage rate was calculated as WT HeLa cells as 1. Solid bars indicate the means, and dots indicate the data from three independent experiments. Data were statistically analyzed using Tukey’s multiple comparisons test. ( F ) Schematic depiction of the difference between the mammalian ULK complex and the yeast Atg1 complex. Mammalian ATG13 binds to two FIP200s within the same FIP200 dimer, contributing to the stability of one ULK complex. Conversely, budding yeast Atg13 binds to two Atg17s within a different Atg17 dimer, allowing for endlessly repeated Atg13–Atg17 interactions. ATG101 in the ULK complex and Atg31-29 in the Atg1 complex are omitted for simplicity. ATG13/Atg13 is shown in yellow, ULK1/Atg1 in magenta, and FIP200/Atg17 in green. Black lines represent interactions. Figure 4—source data 1. PDF file containing original western blots for . Figure 4—source data 2. Original files for western blot analysis displayed in . Figure 4—source data 3. Values used for preparation of the graph in .

Figure Legend Snippet: ( A ) Schematic representation of the CRISPR–Cas9-mediated KI strategy of ATG13 mutations with FLAG tag. The C-terminally FLAG-tagged coding sequence after exon 14 of ATG13 with or without FIP3A, ULK2A, or FU5A mutations were knocked in exon 14 of the Homo sapiens ATG13 locus. As the KI cassette expresses NeoR under the hPGK1 promoter, clones that were successfully knocked in were selected by G418. Cas9-gRNA-targeted sites in the exon 14 of H. sapiens ATG13 locus are displayed in dark blue. The homology arm for KI is presented in magenta, and the ATG13 CDS and mutations in red and cyan, respectively. NeoR is displayed in brown. Scale bar, 0.5 kilobase pair (kb). ( B ) Immunoblot of ATG13-FLAG KI cell lines. WT, ATG13 KO, and indicated KI HeLa cells were lysed, and indicated proteins were detected by immunoblotting using anti-FIP200, anti-ULK1, and anti-FLAG antibodies. ( C ) Colocalization of endogenous levels of ATG13-FLAG mutants with FIP200. Indicated KI cell lines were cultured in the starvation medium for 1 hr and immunostained with anti-FLAG and anti-FIP200 antibodies. Scale bar, 10 μm. ( D ) Halo-LC3 processing assay of ATG13-FLAG KI cell lines. WT, ATG13 KO and KI HeLa cell lines were labeled for 15 min with 100 nm tetramethylrhodamine (TMR)-conjugated Halo ligand and incubated in starvation medium for 1 hr. Cell lysates were subjected to in-gel fluorescence detection. ( E ) Halo processing rate in ( D ). The band intensity of processed Halo and Halo-LC3 in each cell line was quantified, and the relative cleavage rate was calculated as WT HeLa cells as 1. Solid bars indicate the means, and dots indicate the data from three independent experiments. Data were statistically analyzed using Tukey’s multiple comparisons test. ( F ) Schematic depiction of the difference between the mammalian ULK complex and the yeast Atg1 complex. Mammalian ATG13 binds to two FIP200s within the same FIP200 dimer, contributing to the stability of one ULK complex. Conversely, budding yeast Atg13 binds to two Atg17s within a different Atg17 dimer, allowing for endlessly repeated Atg13–Atg17 interactions. ATG101 in the ULK complex and Atg31-29 in the Atg1 complex are omitted for simplicity. ATG13/Atg13 is shown in yellow, ULK1/Atg1 in magenta, and FIP200/Atg17 in green. Black lines represent interactions. Figure 4—source data 1. PDF file containing original western blots for . Figure 4—source data 2. Original files for western blot analysis displayed in . Figure 4—source data 3. Values used for preparation of the graph in .

Techniques Used: CRISPR, FLAG-tag, Sequencing, Clone Assay, Western Blot, Cell Culture, Labeling, Incubation, Fluorescence

( A ) AlphaFold2 model of the full-length ULK1–ATG13 complex. ( B ) AlphaFold2 model of the full-length ATG13 complexed with the homodimer of FIP200 (1–634). ( C ) Predicted aligned error (PAE) plot of ( A ) (left), ( B ) (middle), and ( D ) (right). ( D ) Structure of the ULK1–ATG13–FIP200 core complex with flexible loops. ( E ) The structure in ( D ), color-coded by pLDDT values. ( F ) Cryo-EM structures of the ULK1–ATG13–FIP200 core complex.

Figure Legend Snippet: ( A ) AlphaFold2 model of the full-length ULK1–ATG13 complex. ( B ) AlphaFold2 model of the full-length ATG13 complexed with the homodimer of FIP200 (1–634). ( C ) Predicted aligned error (PAE) plot of ( A ) (left), ( B ) (middle), and ( D ) (right). ( D ) Structure of the ULK1–ATG13–FIP200 core complex with flexible loops. ( E ) The structure in ( D ), color-coded by pLDDT values. ( F ) Cryo-EM structures of the ULK1–ATG13–FIP200 core complex.

Techniques Used: Cryo-EM Sample Prep

Image Search Results

Journal: eLife

Article Title: The triad interaction of ULK1, ATG13, and FIP200 is required for ULK complex formation and autophagy

doi: 10.7554/eLife.101531

Figure Lengend Snippet: ( A ) Structure of the ULK1–FIP200 moiety of the ULK1–ATG13–FIP200 core complex in . The right panel represents the surface model of FIP200 with coloring based on the electrostatic potentials (blue and red indicate positive and negative potentials, respectively). Dotted squares indicate the regions displayed in ( B ). ( B ) Close-up view of the interactions between ULK1 MIT1 and FIP200 (top) and between ULK1 MIT2 and FIP200 (bottom). Left and right indicate AlphaFold2 and cryo-EM (PDB 8SOI) models. ( C ) In vitro pull-down assay between GST-ULK1 (636–1050 aa) WT or FIP2A mutant with MBP-FIP200 (1–634 aa). ( D ) Relative amounts of precipitated MBP-FIP200 in ( C ) were calculated. Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed using Tukey’s multiple comparisons test. ( E ) Effect of the ULK1 FIP2A mutation on the FIP200 interaction in vivo. Ulk1,2 DKO mouse embryonic fibroblasts (MEFs) stably expressing FLAG-tagged ULK1 WT or FIP2A mutant were immunoprecipitated with an anti-FLAG antibody and detected with anti-FIP200, anti-ATG13, and anti-FLAG antibodies. ( F ) Relative amounts of precipitated FIP200 (left) and ATG13 (right) in ( E ) were calculated. Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed using Tukey’s multiple comparisons test. ( G ) Halo-LC3 processing assay of ULK1 FIP2A-expressing cells. Ulk1,2 DKO MEFs stably expressing Halo-LC3 and FLAG-tagged ULK1 WT or FIP2A mutant were labeled for 15 min with 100 nm tetramethylrhodamine (TMR)-conjugated Halo ligand and incubated in starvation medium for 1 hr. Cell lysates were subjected to in-gel fluorescence detection. ( H ) Halo processing rate in ( G ). The band intensity of processed Halo and Halo-LC3 in each cell line was quantified, and the relative cleavage rate was calculated as FLAG-ULK1 WT-expressing cells as 1. Solid bars indicate the means, and dots indicate the data from three independent experiments. Data were statistically analyzed using Tukey’s multiple comparisons test. ( I ) Colocalization of FLAG-ULK1 WT or FIP2A mutant with FIP200. Ulk1,2 DKO MEFs stably expressing FLAG-tagged ULK1 WT or FIP2A mutant were immunostained with anti-FLAG and anti-FIP200 antibodies. Scale bar, 10 μm. Figure 3—source data 1. PDF file containing original western blots or SDS–PAGE for . Figure 3—source data 2. Original files for western blot or SDS–PAGE analysis displayed in . Figure 3—source data 3. Values used for preparation of the graph in .

Article Snippet: Rabbit polyclonal antibodies against FIP200 (17250-1-AP; ProteinTech) and mouse monoclonal antibodies against FLAG (F1804; Sigma-Aldrich) HA (M180-3, MBL) and guinea pig antibody against p62 (GP62-C; PROGEN) were used as primary antibodies for immunostaining.

Techniques: Cryo-EM Sample Prep, In Vitro, Pull Down Assay, Mutagenesis, In Vivo, Stable Transfection, Expressing, Immunoprecipitation, Labeling, Incubation, Fluorescence, Western Blot, SDS Page

Journal: eLife

Article Title: The triad interaction of ULK1, ATG13, and FIP200 is required for ULK complex formation and autophagy

doi: 10.7554/eLife.101531

Figure Lengend Snippet: ( A ) AlphaFold2 model of the ULK1–ATG13 moiety of the ULK1–ATG13–FIP200 core complex in (left), Cryo-EM structure of the ULK1–ATG13 moiety of the ULK1–ATG13–FIP200 core complex (PDB 8SOI), and crystal structure of the yeast Atg1–Atg13 complex (right, PDB 4P1N). ( B ) Close-up view of the interactions between ATG13 MIM(N) and ULK1 MIT2 and between ATG13 MIM(C) and ULK1 MIT1 (right). ( C ) Isothermal titration calorimetry (ITC) results obtained by titration of MBP-ULK1 (636–1050 aa) into a solution of WT or ULK2A mutant of MBP-ATG13 (363–517 aa). Due to weak binding, the K D value for the ULK2A mutant was not accurately determined. ( D ) Effect of the ATG13–FIP3A mutation on endogenous ULK1 levels in vivo. WT or ATG13 KO HeLa cells stably expressing FLAG-tagged ATG13 WT or ULK2A mutant were lysed, and indicated proteins were detected by immunoblotting using anti-FIP200, anti-ULK1, and anti-FLAG antibodies. ( E ) Relative amounts of ULK1 in ( D ) were normalized with β-actin and calculated. Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed using Tukey’s multiple comparisons test. Figure 2—source data 1. PDF file containing original western blots for . Figure 2—source data 2. Original files for western blot analysis displayed in . Figure 2—source data 3. Values used for preparation of the graph in .

Techniques: Cryo-EM Sample Prep, Isothermal Titration Calorimetry, Titration, Mutagenesis, Binding Assay, In Vivo, Stable Transfection, Expressing, Western Blot

Journal: eLife

Article Title: The triad interaction of ULK1, ATG13, and FIP200 is required for ULK complex formation and autophagy

doi: 10.7554/eLife.101531

Figure Lengend Snippet: ( A ) Domain architecture of ULK1, ATG13, and FIP200. Regions used for the AlphaFold2 complex prediction are underlined. ( B ) Structure of the ULK1–ATG13–FIP200 core complex predicted by AlphaFold2. Flexible loop regions in FIP200 were removed from the figure for clarity. N and C indicate N- and C-terminal regions, respectively. ( C ) Close-up view of the interactions between ATG13 and FIP200. The bottom panels represent the surface model of FIP200 with the coloring based on the electrostatic potentials (blue and red indicate positive and negative potentials, respectively). ( D ) Isothermal titration calorimetry (ITC) results obtained by titration of MBP-ATG13 (363–517 aa) WT or FIP3A mutant into an FIP200 (1–634 aa) solution. ( E ) Effect of the ATG13 FIP3A mutation on the FIP200 interaction in vivo. ATG13 KO HeLa cells stably expressing FLAG-tagged ATG13 WT or FIP3A were immunoprecipitated with an anti-FLAG antibody and detected with anti-FIP200, anti-ULK1, and anti-FLAG antibodies. ( F ) Relative amounts of precipitated FIP200 in ( E ) were calculated. Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed using Tukey’s multiple comparisons test. Figure 1—source data 1. PDF file containing original western blots for . Figure 1—source data 2. Original files for western blot analysis displayed in . Figure 1—source data 3. Values used for preparation of the graph in .

Techniques: Isothermal Titration Calorimetry, Titration, Mutagenesis, In Vivo, Stable Transfection, Expressing, Immunoprecipitation, Western Blot

Journal: eLife

Article Title: The triad interaction of ULK1, ATG13, and FIP200 is required for ULK complex formation and autophagy

doi: 10.7554/eLife.101531

Figure Lengend Snippet: ( A ) Comparison of ATG13 expression level. WT, ATG13 KO stably expressing ATG13-FLAG, and ATG13-FLAG KI HeLa cells were lysed, and indicated proteins were detected by immunoblotting using anti-ATG13, anti-FIP200, anti-ULK1, and anti-β-actin antibodies. ( B ) Relative amounts of ATG13 in ( A ) were calculated as WT HeLa cells as 1. Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed using Tukey’s multiple comparisons test. ( C ) Colocalization of ATG9A and FIP200 in ATG13-FLAG KI cells. Indicated KI cell lines expressing ATG9A-HA were cultured in the starvation medium for 1 hr and immunostained with anti-FLAG, anti-FIP200, and anti-p62 antibodies. Scale bar, 10 μm. ( D ) ULK1-dependent phosphorylation of ATG14 in ATG13-FLAG KI cell lines. WT, ATG13 KO and KI HeLa cell lines cultured in the starvation medium for 1 hr. Indicated proteins were detected by immunoblotting using anti-ATG14 phospho-S29, anti-ATG14, and anti-β-actin antibodies. ( E ) ATG14 phosphorylation rate in ( D ). The band intensity of p-ATG14 and ATG14 in each cell line was quantified, and the phosphorylation rate was calculated as WT HeLa cells as 1. Solid bars indicate the means, and dots indicate the data from three independent experiments. Data were statistically analyzed using Tukey’ s multiple comparisons test. Figure 4—figure supplement 1—source data 1. PDF file containing original western blots for . Figure 4—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 4—figure supplement 1—source data 3. Values used for preparation of the graph in .

Techniques: Comparison, Expressing, Stable Transfection, Western Blot, Cell Culture, Phospho-proteomics

Journal: eLife

Article Title: The triad interaction of ULK1, ATG13, and FIP200 is required for ULK complex formation and autophagy

doi: 10.7554/eLife.101531

Figure Lengend Snippet: ( A ) Schematic representation of the CRISPR–Cas9-mediated KI strategy of ATG13 mutations with FLAG tag. The C-terminally FLAG-tagged coding sequence after exon 14 of ATG13 with or without FIP3A, ULK2A, or FU5A mutations were knocked in exon 14 of the Homo sapiens ATG13 locus. As the KI cassette expresses NeoR under the hPGK1 promoter, clones that were successfully knocked in were selected by G418. Cas9-gRNA-targeted sites in the exon 14 of H. sapiens ATG13 locus are displayed in dark blue. The homology arm for KI is presented in magenta, and the ATG13 CDS and mutations in red and cyan, respectively. NeoR is displayed in brown. Scale bar, 0.5 kilobase pair (kb). ( B ) Immunoblot of ATG13-FLAG KI cell lines. WT, ATG13 KO, and indicated KI HeLa cells were lysed, and indicated proteins were detected by immunoblotting using anti-FIP200, anti-ULK1, and anti-FLAG antibodies. ( C ) Colocalization of endogenous levels of ATG13-FLAG mutants with FIP200. Indicated KI cell lines were cultured in the starvation medium for 1 hr and immunostained with anti-FLAG and anti-FIP200 antibodies. Scale bar, 10 μm. ( D ) Halo-LC3 processing assay of ATG13-FLAG KI cell lines. WT, ATG13 KO and KI HeLa cell lines were labeled for 15 min with 100 nm tetramethylrhodamine (TMR)-conjugated Halo ligand and incubated in starvation medium for 1 hr. Cell lysates were subjected to in-gel fluorescence detection. ( E ) Halo processing rate in ( D ). The band intensity of processed Halo and Halo-LC3 in each cell line was quantified, and the relative cleavage rate was calculated as WT HeLa cells as 1. Solid bars indicate the means, and dots indicate the data from three independent experiments. Data were statistically analyzed using Tukey’s multiple comparisons test. ( F ) Schematic depiction of the difference between the mammalian ULK complex and the yeast Atg1 complex. Mammalian ATG13 binds to two FIP200s within the same FIP200 dimer, contributing to the stability of one ULK complex. Conversely, budding yeast Atg13 binds to two Atg17s within a different Atg17 dimer, allowing for endlessly repeated Atg13–Atg17 interactions. ATG101 in the ULK complex and Atg31-29 in the Atg1 complex are omitted for simplicity. ATG13/Atg13 is shown in yellow, ULK1/Atg1 in magenta, and FIP200/Atg17 in green. Black lines represent interactions. Figure 4—source data 1. PDF file containing original western blots for . Figure 4—source data 2. Original files for western blot analysis displayed in . Figure 4—source data 3. Values used for preparation of the graph in .

Techniques: CRISPR, FLAG-tag, Sequencing, Clone Assay, Western Blot, Cell Culture, Labeling, Incubation, Fluorescence

Journal: eLife

Article Title: The triad interaction of ULK1, ATG13, and FIP200 is required for ULK complex formation and autophagy

doi: 10.7554/eLife.101531

Figure Lengend Snippet: ( A ) AlphaFold2 model of the full-length ULK1–ATG13 complex. ( B ) AlphaFold2 model of the full-length ATG13 complexed with the homodimer of FIP200 (1–634). ( C ) Predicted aligned error (PAE) plot of ( A ) (left), ( B ) (middle), and ( D ) (right). ( D ) Structure of the ULK1–ATG13–FIP200 core complex with flexible loops. ( E ) The structure in ( D ), color-coded by pLDDT values. ( F ) Cryo-EM structures of the ULK1–ATG13–FIP200 core complex.

Techniques: Cryo-EM Sample Prep

Fig. 2 NEK9 is degraded by selective autophagy. a Immunoﬂuorescence microscopy of MEFs expressing GFP-NEK9 and mRuby3-GABARAP under nutrient-rich conditions and amino acid and serum starvation (2 h) conditions with or without 100 nM baﬁlomycin A1 (baf A1). b Quantiﬁcation of the number of NEK9 puncta in (a); p values correspond to a Tukey’s multiple comparisons test. c Immunoﬂuorescence microscopy of wild-type and Fip200-KO MEFs expressing GFP-NEK9 (top), and wild-type MEFs expressing GFP-NEK9 W967A (LIR-mutant) (bottom) after starvation (2 h). d, e Quantiﬁcation of the number of NEK9 puncta in (c); p values correspond to two-tailed Mann–Whitney tests. f Wild-type and Fip200-KO MEFs were incubated under starvation conditions with or without 100 nM baﬁlomycin A1 for the indicated time. Whole-cell lysates were subjected to immunoblotting. g Quantiﬁcation of the intensity of the NEK9 bands in (f). Data represent the mean ± SEM values of three independent experiments. h Immunoblotting of indicated organs of three-month-old Atg5+/+ (WT) and Atg5−/−;NSE-Atg5 (KO) mice. Data are representative of three biologically independent replicates. For (b, d, and e), data were collected from 100 cells for each condition. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 10th to 90th percentile. Scale bars, 10 µm and 3 µm (insets).

Journal: Nature communications

Article Title: NEK9 regulates primary cilia formation by acting as a selective autophagy adaptor for MYH9/myosin IIA.

doi: 10.1038/s41467-021-23599-7

Figure Lengend Snippet: Fig. 2 NEK9 is degraded by selective autophagy. a Immunoﬂuorescence microscopy of MEFs expressing GFP-NEK9 and mRuby3-GABARAP under nutrient-rich conditions and amino acid and serum starvation (2 h) conditions with or without 100 nM baﬁlomycin A1 (baf A1). b Quantiﬁcation of the number of NEK9 puncta in (a); p values correspond to a Tukey’s multiple comparisons test. c Immunoﬂuorescence microscopy of wild-type and Fip200-KO MEFs expressing GFP-NEK9 (top), and wild-type MEFs expressing GFP-NEK9 W967A (LIR-mutant) (bottom) after starvation (2 h). d, e Quantiﬁcation of the number of NEK9 puncta in (c); p values correspond to two-tailed Mann–Whitney tests. f Wild-type and Fip200-KO MEFs were incubated under starvation conditions with or without 100 nM baﬁlomycin A1 for the indicated time. Whole-cell lysates were subjected to immunoblotting. g Quantiﬁcation of the intensity of the NEK9 bands in (f). Data represent the mean ± SEM values of three independent experiments. h Immunoblotting of indicated organs of three-month-old Atg5+/+ (WT) and Atg5−/−;NSE-Atg5 (KO) mice. Data are representative of three biologically independent replicates. For (b, d, and e), data were collected from 100 cells for each condition. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 10th to 90th percentile. Scale bars, 10 µm and 3 µm (insets).

Article Snippet: The following antibodies were used for immunocytochemistry and immunohistochemistry: rabbit polyclonal antibodies against FIP200 (1:200 dilution, 17250-1-AP; ProteinTech), WIPI2 (1:200 dilution, SAB4200400; Sigma-Aldrich), LAMP1 (1:200 dilution, ab24170; Abcam), NEK9 (1:200 dilution, A301-139A; Bethyl), OFD1 (1:100 dilution, NBP1-89355; Novus Biologicals), and pericentrin (1:200 dilution, abcam; 4448) and mouse monoclonal antibodies against LC3 (1:100 dilution, CTB-LC3-2-IC; CosmoBio), NEK9 (1:100 dilution, sc-100401; Santa Cruz), FIP200 (1:200 dilution, MABC128; Sigma-Aldrich), WIPI2 (1:200 dilution, MABC91; Sigma-Aldrich), LAMP1 (1:200 dilution, ab25630; abcam), and ARL13B (1:200 dilution, ab136648; abcam).

Techniques: Microscopy, Expressing, Mutagenesis, Two Tailed Test, MANN-WHITNEY, Incubation, Western Blot

Fig. 3 Selective autophagy of NEK9 is required for primary cilia formation. a Generation of homozygous Nek9W967A (LIR-mutant) cell lines by CRISPR- mediated recombination using a donor plasmid harboring short homology arms. b Immunoblotting of wild-type or Nek9W967A MEFs (two independent clones, #7 and #13) cultured in nutrient-rich medium. c, Quantiﬁcation of the intensity of the NEK9 bands in (b). Data represent the mean ± SEM of three independent experiments. d Immunoﬂuorescence microscopy of wild-type or Nek9W967A MEFs after serum starvation (24 h). Centrosomes and primary cilia were stained with anti-pericentrin and anti-ARL13B antibodies, respectively. e The frequency of ciliated cells in (d). f Quantiﬁcation of cilia length in (d). Data were collected from 100 ciliated cells for each cell-type. g Immunoﬂuorescence microscopy of wild-type or Fip200-KO MEFs after serum starvation (24 h). h The frequency of ciliated cells in (g), as in (e). i Quantiﬁcation of cilia length in (g), as in (f). Data were collected from 100 ciliated cells for each cell-type. j Immunoﬂuorescence microscopy of wild-type or Nek9-KO MEFs stably expressing the indicated constructs. k The frequency of ciliated cells in (j), as in (e). l Quantiﬁcation of cilia length in (j), as in (f). Data were collected from 100 ciliated cells for each cell-type. p values correspond to Tukey’s multiple comparisons tests in (c, e, f, k, and l) and two-tailed Mann–Whitney tests in (h) and (i); *p < 0.0001. Scale bars, 10 µm and 3 µm (insets). Data represent the mean ± SEM of ﬁve independent experiments (300 cells were counted in each experiment) in (e, h, k). Solid bars indicate the medians, boxes the interquartile range (25th–75th percentile), and whiskers the 10th–90th percentile in (f, i, and l).

Journal: Nature communications

Article Title: NEK9 regulates primary cilia formation by acting as a selective autophagy adaptor for MYH9/myosin IIA.

doi: 10.1038/s41467-021-23599-7

Figure Lengend Snippet: Fig. 3 Selective autophagy of NEK9 is required for primary cilia formation. a Generation of homozygous Nek9W967A (LIR-mutant) cell lines by CRISPR- mediated recombination using a donor plasmid harboring short homology arms. b Immunoblotting of wild-type or Nek9W967A MEFs (two independent clones, #7 and #13) cultured in nutrient-rich medium. c, Quantiﬁcation of the intensity of the NEK9 bands in (b). Data represent the mean ± SEM of three independent experiments. d Immunoﬂuorescence microscopy of wild-type or Nek9W967A MEFs after serum starvation (24 h). Centrosomes and primary cilia were stained with anti-pericentrin and anti-ARL13B antibodies, respectively. e The frequency of ciliated cells in (d). f Quantiﬁcation of cilia length in (d). Data were collected from 100 ciliated cells for each cell-type. g Immunoﬂuorescence microscopy of wild-type or Fip200-KO MEFs after serum starvation (24 h). h The frequency of ciliated cells in (g), as in (e). i Quantiﬁcation of cilia length in (g), as in (f). Data were collected from 100 ciliated cells for each cell-type. j Immunoﬂuorescence microscopy of wild-type or Nek9-KO MEFs stably expressing the indicated constructs. k The frequency of ciliated cells in (j), as in (e). l Quantiﬁcation of cilia length in (j), as in (f). Data were collected from 100 ciliated cells for each cell-type. p values correspond to Tukey’s multiple comparisons tests in (c, e, f, k, and l) and two-tailed Mann–Whitney tests in (h) and (i); *p < 0.0001. Scale bars, 10 µm and 3 µm (insets). Data represent the mean ± SEM of ﬁve independent experiments (300 cells were counted in each experiment) in (e, h, k). Solid bars indicate the medians, boxes the interquartile range (25th–75th percentile), and whiskers the 10th–90th percentile in (f, i, and l).

Techniques: Mutagenesis, CRISPR, Plasmid Preparation, Western Blot, Clone Assay, Cell Culture, Microscopy, Staining, Stable Transfection, Expressing, Construct, Two Tailed Test, MANN-WHITNEY

CRISPR-mediated genome-wide screen using an autophagic flux reporter. (A) Schematic representation of the autophagic flux reporter GFP-LC3-RFP. GFP-LC3-RFP is cleaved by endogenous ATG4 family proteins to yield equimolar amounts of GFP-LC3 (autophagy substrate) and RFP (internal control). Reduction in the GFP:RFP ratio indicates autophagic activity. (B) HEK293T cells expressing Cas9 and GFP-LC3-RFP were transduced with or without sgRNAs targeting ATG9A and FIP200 and selected with puromycin. The GFP and RFP intensities were determined by flow cytometry under nutrient-rich and starvation conditions. The autophagy-deficient population is indicated by the region of interest (ROI). (C) Schematic representation of the CRISPR-mediated genome-wide screen. An sgRNA library (GeCKO) was introduced to HEK293T cells expressing Cas9 and GFP-LC3-RFP. The cell population that did not respond to starvation (indicated by the ROI) was collected by FACS and expanded. After repeating this enrichment process three times, genomic DNA was extracted and subjected to next-generation sequencing. The proportion (%) of the autophagy-deficient population is indicated by the ROI. (D) Scatterplot of the results of two replicates. Data represent log2 (fold change) of read counts of individual sgRNAs before versus after enrichment. Enriched sgRNAs are shown in the separate panel. Canonical ATG genes and known autophagy-related genes (green), genes encoding HOPS and ESCRT components (blue), negative regulators of mTORC1 (yellow), and high-scoring genes not previously linked to autophagy (magenta) are indicated.

Journal: The Journal of Cell Biology

Article Title: Genome-wide CRISPR screen identifies TMEM41B as a gene required for autophagosome formation

doi: 10.1083/jcb.201804132

Figure Lengend Snippet: CRISPR-mediated genome-wide screen using an autophagic flux reporter. (A) Schematic representation of the autophagic flux reporter GFP-LC3-RFP. GFP-LC3-RFP is cleaved by endogenous ATG4 family proteins to yield equimolar amounts of GFP-LC3 (autophagy substrate) and RFP (internal control). Reduction in the GFP:RFP ratio indicates autophagic activity. (B) HEK293T cells expressing Cas9 and GFP-LC3-RFP were transduced with or without sgRNAs targeting ATG9A and FIP200 and selected with puromycin. The GFP and RFP intensities were determined by flow cytometry under nutrient-rich and starvation conditions. The autophagy-deficient population is indicated by the region of interest (ROI). (C) Schematic representation of the CRISPR-mediated genome-wide screen. An sgRNA library (GeCKO) was introduced to HEK293T cells expressing Cas9 and GFP-LC3-RFP. The cell population that did not respond to starvation (indicated by the ROI) was collected by FACS and expanded. After repeating this enrichment process three times, genomic DNA was extracted and subjected to next-generation sequencing. The proportion (%) of the autophagy-deficient population is indicated by the ROI. (D) Scatterplot of the results of two replicates. Data represent log2 (fold change) of read counts of individual sgRNAs before versus after enrichment. Enriched sgRNAs are shown in the separate panel. Canonical ATG genes and known autophagy-related genes (green), genes encoding HOPS and ESCRT components (blue), negative regulators of mTORC1 (yellow), and high-scoring genes not previously linked to autophagy (magenta) are indicated.

Article Snippet: The following antibodies were used for immunocytochemistry: rabbit polyclonal antibody against FIP200 (17250-1-AP; ProteinTech), rabbit polyclonal antibody against WIPI2 (SAB4200400; Sigma-Aldrich) and p62/SQSTM1 (PM045; MBL), and mouse monoclonal antibody against LC3 (CTB-LC3-2-IC; CosmoBio).

Techniques: CRISPR, Genome Wide, Control, Activity Assay, Expressing, Transduction, Flow Cytometry, Next-Generation Sequencing

Defective autophagosome formation in TMEM41B-KO and VMP1-KO cells. (A) WT, TMEM41B-KO, VMP1-KO, and rescued TMEM41B-KO HEK293T cells were cultured under nutrient-rich and starvation (Stv) conditions for 2 h and subjected to immunofluorescence microscopy. Bars: 10 µm (main images); 500 nm (insets). (B) Numbers of FIP200 and WIPI2 puncta in cells under nutrient-rich and starvation conditions were quantified as in . Data were collected from >70 cells for each sample. (C) Quantification of colocalization between FIP200 and LC3 and between WIPI2 and LC3. Data were collected from >70 cells for each sample. (D) WT, TMEM41B-KO, VMP1-KO, and rescued TMEM41B-KO cells expressing mRuby3-STX17TM were starved for 2 h and observed by fluorescence microscopy. Data were collected from >40 cells and quantified as in . Bars: 10 µm (main images); 500 nm (insets). (E) Transmission EM of WT, VMP1-KO, and TMEM41B-KO cells under starvation conditions (2 h). Insets indicate an autophagosome (in WT) or ferritin clusters (in KO). Bars: 500 nm (main images); 50 nm (insets). (F) WT, TMEM41B-KO, VMP1-KO, and rescued TMEM41B-KO HEK293T cells were stained with LipidTOX. Bar, 10 µm.

Journal: The Journal of Cell Biology

Article Title: Genome-wide CRISPR screen identifies TMEM41B as a gene required for autophagosome formation

doi: 10.1083/jcb.201804132

Figure Lengend Snippet: Defective autophagosome formation in TMEM41B-KO and VMP1-KO cells. (A) WT, TMEM41B-KO, VMP1-KO, and rescued TMEM41B-KO HEK293T cells were cultured under nutrient-rich and starvation (Stv) conditions for 2 h and subjected to immunofluorescence microscopy. Bars: 10 µm (main images); 500 nm (insets). (B) Numbers of FIP200 and WIPI2 puncta in cells under nutrient-rich and starvation conditions were quantified as in . Data were collected from >70 cells for each sample. (C) Quantification of colocalization between FIP200 and LC3 and between WIPI2 and LC3. Data were collected from >70 cells for each sample. (D) WT, TMEM41B-KO, VMP1-KO, and rescued TMEM41B-KO cells expressing mRuby3-STX17TM were starved for 2 h and observed by fluorescence microscopy. Data were collected from >40 cells and quantified as in . Bars: 10 µm (main images); 500 nm (insets). (E) Transmission EM of WT, VMP1-KO, and TMEM41B-KO cells under starvation conditions (2 h). Insets indicate an autophagosome (in WT) or ferritin clusters (in KO). Bars: 500 nm (main images); 50 nm (insets). (F) WT, TMEM41B-KO, VMP1-KO, and rescued TMEM41B-KO HEK293T cells were stained with LipidTOX. Bar, 10 µm.

Techniques: Cell Culture, Immunofluorescence, Microscopy, Expressing, Fluorescence, Transmission Assay, Staining

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