Journal: Molecular Cell

Article Title: The Cargo Receptor NDP52 Initiates Selective Autophagy by Recruiting the ULK Complex to Cytosol-Invading Bacteria

doi: 10.1016/j.molcel.2019.01.041

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-FIP200 , ProteinTech , Cat# 10043-2-AP; RRID: AB_2253571.

Techniques: Luciferase, Transduction, Virus, Recombinant, Plasmid Preparation, Protease Inhibitor, Reverse Transcription, Negative Control, Software, Chromatography, Molecular Weight

Journal: Cell Death & Disease

Article Title: Transient receptor potential ion channel TRPM2 promotes AML proliferation and survival through modulation of mitochondrial function, ROS, and autophagy

doi: 10.1038/s41419-020-2454-8

Figure Lengend Snippet: a Western blotting was performed to determine expression of HIF-1α, HIF-2α, FOXO3a, IQGAP1, Nrf2, CREB, and CREB phosphorylation in TRPM2-depleted U937 cells. In these experiments, two knockout (KO-1-2) and two scrambled clones (Scr-2-3) were selected randomly for western blots with or without treatment with 0.3 µM doxorubicin. b Expression of autophagy proteins ULK1, Atg7, Atg5, Atg13, FIP200, Atg101, and transcription factor ATF4 was examined by western blotting. p62, Tom20, and LC3B-I and II, which are modified in autophagy, were also studied. Actin was probed to confirm equivalent loading. In a and b , western blots performed for each protein are shown on the left. One blot for each protein is shown here and the others in Supplemental Information Figs. , . Densitometry measurements from two to three experiments for each protein were standardized to results for each experiment’s average untreated scrambled control and means ± s.e.m. calculated and shown on the right ( n = 4-6, Tom20 n = 8). * p ≤ 0.001, ** p < 0.03, group effect, Scr vs KO, two-way ANOVA. c RT-PCR was used to measure TRPM2, HIF-1α, HIF-2α, CREB, ULK1, and Atg7 mRNA in TRPM2-depleted leukemia cells. Results summarizing two (TRPM2, ULK1), three (Atg7) or four (HIF-1/2α, CREB) experiments are shown (mean ± s.e.m., n = 8-16). * p ≤ 0.0001, ** p < 0.015, one-way ANOVA. d U937 cell were incubated with or without bafilomycin A1, and conversion of LCB-I to II was examined with western blotting. Four experiments were performed. One blot is shown here and the others in Supplemental Information Fig. . Densitometry measurements were obtained. Relative autophagic flux was calculated as: (O.D. LC3B-II + Bafilomycin/O.D. Actin) - (O.D. LC3B-II-Bafilomycin/O.D. Actin) for each band. Means ± s.e.m. for the four experiments for each cell line are shown below the figure ( n = 8). * p < 0.05, unpaired, two-tailed t -test. e Western blotting was performed to measure TRPM2, CREB, ATF4, ULK1, Atg7, Atg5, and LC3B-I and II expression in four experiments after TRPM2-L reconstitution. One blot is shown here and the others in Supplemental Information Fig. . Densitometry measurements were normalized to each blots’ untreated scrambled control, and mean densitometry measurements ± s.e.m. for experiments with each protein are shown on the right. * p < 0.04 ( n = 4) one-way ANOVA.

Article Snippet: Blots were probed with anti-TRPM2-C (1:300; Bethyl Laboratories, Montgomery, TX, USA); with antibodies to ATF4 (1:400), Atg7 (1:2000), Atg13 (1:500), Atg101 (1:500), pCREB1 and CREB1 (1:250), FIP200 (1:300), forkhead box transcription factor 3a (FOXO3a; 1:400), HIF-1α (1:250), Nrf2 (1:400), and ULK1 (1:1000) from Cell Signaling Technology (Boston, MA, USA); Atg5 (1:1000) from Medical Biological Lab (Japan); COX6B1 (1:500), MT-CO2 (1:500), MT-ND2 (1:500), and NDUFA13 (1:300) from LSBio (Seattle, WA, USA); HIF-2α (1:500) and LC3B (1:2000) from Novus (Littleton, CO, USA); IQGAP1 (1:5000) from Abcam (Cambridge, MA, USA); p62 (1:5000) from American Research Products (Waltham, MA, USA); Tom20 (1:5000) from Santa Cruz Biotech (Dallas, TX, USA); and actin (1:5000) from Sigma (St. Louis, MO, USA).

Techniques: Western Blot, Expressing, Knock-Out, Clone Assay, Modification, Reverse Transcription Polymerase Chain Reaction, Incubation, Two Tailed Test

Journal: Molecular cell

Article Title: FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates.

doi: 10.1016/j.molcel.2019.01.035

Figure Lengend Snippet: Figure 2. p62 FIR Phosphorylation Enhances FIP200 Binding (A) Overview of phosphosites in the FIR of affinity-purified p62 as identified by mass spectrometry (Figures S2A and S2B). (B) Phospho-mimicking mutations were introduced in GST-p62 FIR as follows: 1P (S349D); 3P (S365D, S366D, S370D); 4P (S349D, S365D, S366D, S370D). GSH beads were coated with GST or the GST-p62 FIRs, incubated with GFP-FIP200 CTR (aa 1458–1594) and imaged by microscopy at equilibrium. GFP signals on the beads were normalized to the signal of GFP-FIP200 CTR bound to GST-p62 FIR 4P. Average intensity and SEM for n = 3 are shown. Significant differences are indicated with * when p value % 0.05, ** when p value % 0.01, and *** when p value % 0.001. The same beads were analyzed by SDS-PAGE like in a pull-down experiment (Figure S2D). (C) The binding of GFP-LC3B to GST-p62 phospho-mimicking mutants was assessed like in (B). (D) GSH beads were coated with full-length GST-FIP200 and incubated with full-length mCherry-p62 WT or the 4P phospho-mimicking mutant. Beads were imaged by microscopy. The mCherry signal is shown in false color (ImageJ: Fire).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-p62 BD Bioscience Cat#610832; RRID: AB_398152 Mouse anti-GST Sigma Cat#SAB4200237; RRID: AB259845 Rabbit anti-FIP200 (D10D11) Cell Signaling Technology Cat#12436 Rabbit anti-FIP200 Atlas antibodies Cat#HPA053049; RRID: AB_2682025 Mouse anti-FIP200 (6B2) MFPL antibody facility N/A Rabbit anti-Stx17 Sigma Cat#HPA001204; RRID: AB_1080118 Mouse anti-LC3B (clone 2G6) nanoTools Cat#0260-100 Rabbit anti-GABARAP Cell Signaling Technology Cat#13733 Mouse anti-GFP Roche Cat#11814460001; RRID: AB_390913 Mouse anti-myc (clone 4A6) Millipore Cat#05-724; RRID: AB_310809 Rabbit anti-Atg19 Eurogentec N/A Rabbit anti-Ape1 Gift from Kraft lab (Institute for Biochem.

Techniques: Phospho-proteomics, Binding Assay, Mass Spectrometry, Incubation, Microscopy, SDS Page, Mutagenesis

Journal: Molecular cell

Article Title: FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates.

doi: 10.1016/j.molcel.2019.01.035

Figure Lengend Snippet: Figure 3. LC3B Competes with FIP200 for p62 Binding, and FIP200 Is Excluded from the Autophagosomal Lumen (A) RFP trap beads were coated with mCherry-p62 FIR 4P and incubated with the GFP-FIP200 CTR (aa 1458-1594, 1.1 mM). Then, increasing concentrations of LC3B were added. Beads were imaged by microscopy. The intensities of the GFP and mCherry signals on the beads were measured and plotted against the LC3B concentrations (lower panels). A zoom-in of the plot at the lowest LC3B concentrations is shown on the left. Average intensities and SD for n = 3 are shown. Negative and positive controls of binding are shown in Figure S3B. (B) HeLa cells were left untreated, starved or treated with puromycin both in presence or absence of bafilomycin. Cell lysates were then treated with proteinase K in the presence or absence of Triton X-100 and analyzed by western blotting with anti-p62 and anti-STX17. LC3B processing was used to monitor autophagy induction. The percentage of protease protection for FIP200, in comparison to p62 and STX17 is plotted on the right. Average protection and SD for n = 3 are shown. Protease protection in starved cells treated with wortmannin and/or bafilomycin are shown in Figure S3D. See also Figure S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-p62 BD Bioscience Cat#610832; RRID: AB_398152 Mouse anti-GST Sigma Cat#SAB4200237; RRID: AB259845 Rabbit anti-FIP200 (D10D11) Cell Signaling Technology Cat#12436 Rabbit anti-FIP200 Atlas antibodies Cat#HPA053049; RRID: AB_2682025 Mouse anti-FIP200 (6B2) MFPL antibody facility N/A Rabbit anti-Stx17 Sigma Cat#HPA001204; RRID: AB_1080118 Mouse anti-LC3B (clone 2G6) nanoTools Cat#0260-100 Rabbit anti-GABARAP Cell Signaling Technology Cat#13733 Mouse anti-GFP Roche Cat#11814460001; RRID: AB_390913 Mouse anti-myc (clone 4A6) Millipore Cat#05-724; RRID: AB_310809 Rabbit anti-Atg19 Eurogentec N/A Rabbit anti-Ape1 Gift from Kraft lab (Institute for Biochem.

Techniques: Binding Assay, Incubation, Microscopy, Western Blot, Comparison

Journal: Molecular cell

Article Title: FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates.

doi: 10.1016/j.molcel.2019.01.035

Figure Lengend Snippet: Figure 4. Crystal Structure of FIP200 CTR (A) Crystal structure of the FIP200 CTR monomer. The molecule comprises a helix, a linker, and a globular Claw domain. The structure is colored according to its secondary structure elements (helix, purple; b strands, orange; loops, gray). (B) Close-up of the Claw domain. The fold resembles a Claw with the b sheet being the palm of the Claw and loops L2, L4, and L5 flexed fingers. Same view and coloring used in (A). (C) Topology plot of monomeric FIP200 CTR. a helices are shown as purple cylinders and b strands as orange arrows. (D) Dimerization of FIP200 CTR. The homodimer is formed via two interfaces: interface 1 (blue box) and interface 2 (red box). Monomers are colored in orange and green. Interface residues are shown as sticks and labeled for only one monomer. (E) Surface conservation plot of the FIP200 CTR monomer based on the sequence alignment of 11 different species (Figure S5B). Conserved residues are colored in purple and non-conserved residues in cyan. The second monomer is shown in cartoon representation. (F) Analytical size-exclusion chromatography coupled to right-angle light scattering. Absorption at 280 nm (blue) and oligomeric state (as apparent molecular weight, divided by molecular weight of the monomer) (red) are plotted against the retention volume (mL). See also Figures S4 and S5.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-p62 BD Bioscience Cat#610832; RRID: AB_398152 Mouse anti-GST Sigma Cat#SAB4200237; RRID: AB259845 Rabbit anti-FIP200 (D10D11) Cell Signaling Technology Cat#12436 Rabbit anti-FIP200 Atlas antibodies Cat#HPA053049; RRID: AB_2682025 Mouse anti-FIP200 (6B2) MFPL antibody facility N/A Rabbit anti-Stx17 Sigma Cat#HPA001204; RRID: AB_1080118 Mouse anti-LC3B (clone 2G6) nanoTools Cat#0260-100 Rabbit anti-GABARAP Cell Signaling Technology Cat#13733 Mouse anti-GFP Roche Cat#11814460001; RRID: AB_390913 Mouse anti-myc (clone 4A6) Millipore Cat#05-724; RRID: AB_310809 Rabbit anti-Atg19 Eurogentec N/A Rabbit anti-Ape1 Gift from Kraft lab (Institute for Biochem.

Techniques: Labeling, Sequencing, Size-exclusion Chromatography, Molecular Weight

Journal: Molecular cell

Article Title: FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates.

doi: 10.1016/j.molcel.2019.01.035

Figure Lengend Snippet: Figure 5. p62 LIR Motif Binding Depends on a Positively Charged Pocket in FIP200 CTR (A) Electrostatic surface potential of the FIP200 Claw domain. Positively and negatively charged surfaces are colored in blue and red, respectively. The coor- dination of sulfate ions and amino acids of interest are shown as sticks. (B) GSH beads were coated with GST-p62 FIR 4P, incubated with the indicated GFP-FIP200 CTR (aa 1458–1594) mutants and imaged by microscopy. For each sample the GFP intensity was normalized to the signal of GFP-FIP200 CTR WT on GST-p62 FIR 4P-coated beads. Average intensity and SEM for n = 3 are shown. Significant differences are indicated with * when p value % 0.05, ** when p value % 0.01, and *** when p value % 0.001. Protein inputs are shown in Figure S5C. (C) mCherry-p62 (2 mM) was incubated with GST-4x ubiquitin (10 mM) to form condensates in solution. Pre-formed condensates were incubated with 1 mM GFP- FIP200 CTR (aa 1458–1594). The recruitment of GFP-FIP200 CTR to p62-ubiquitin clusters was monitored by confocal microscopy. Scale bar, 5 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-p62 BD Bioscience Cat#610832; RRID: AB_398152 Mouse anti-GST Sigma Cat#SAB4200237; RRID: AB259845 Rabbit anti-FIP200 (D10D11) Cell Signaling Technology Cat#12436 Rabbit anti-FIP200 Atlas antibodies Cat#HPA053049; RRID: AB_2682025 Mouse anti-FIP200 (6B2) MFPL antibody facility N/A Rabbit anti-Stx17 Sigma Cat#HPA001204; RRID: AB_1080118 Mouse anti-LC3B (clone 2G6) nanoTools Cat#0260-100 Rabbit anti-GABARAP Cell Signaling Technology Cat#13733 Mouse anti-GFP Roche Cat#11814460001; RRID: AB_390913 Mouse anti-myc (clone 4A6) Millipore Cat#05-724; RRID: AB_310809 Rabbit anti-Atg19 Eurogentec N/A Rabbit anti-Ape1 Gift from Kraft lab (Institute for Biochem.

Techniques: Binding Assay, Incubation, Microscopy, Ubiquitin Proteomics, Confocal Microscopy

Journal: Molecular cell

Article Title: FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates.

doi: 10.1016/j.molcel.2019.01.035

Figure Lengend Snippet: Figure 6. p62-FIP200 Interaction in Cells (A) Colocalization analysis of p62 and FIP200 in HAP1 cells (WT or ATG7KO) left untreated or treated with wortmannin (1 mM) for 1 h. Endogenous p62 and FIP200 were detected by immunofluorescence. Scale bar, 10 mm. Average percentages of colocalization and SEM for n = 3 are shown. Significant differences are indicated with * when p value % 0.05, ** when p value % 0.01, and *** when p value % 0.001. (B) Immunofluorescence of p62 and FIP200 in HAP1 STG-p62 cell line left untreated or treated with bafilomycin (400 nM) for 1 h. p62 was detected through the GFP tag fused to the endogenous protein and FIP200 was detected by immunofluorescence. Scale bar, 10 mm. Average percentages of colocalization and SEM (n = 2) are shown. Significant differences are indicated with * when p value % 0.05, ** when p value % 0.01, and *** when p value % 0.001. (C) The C terminus of endogenous FIP200 was tagged in HAP1 cells with GFP-TEV-Strep (FIP200-STG). Affinity purification was performed using HAP1 WT or FIP200-STG cells and the bound material was analyzed by western blotting with anti-p62. The intensities of the p62 bands were normalized for the total level of p62 in the lysate (input). Average p62 levels and SD for n = 4 are shown. Three additional replicates of the immunoprecipitation are shown in Figure S6C.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-p62 BD Bioscience Cat#610832; RRID: AB_398152 Mouse anti-GST Sigma Cat#SAB4200237; RRID: AB259845 Rabbit anti-FIP200 (D10D11) Cell Signaling Technology Cat#12436 Rabbit anti-FIP200 Atlas antibodies Cat#HPA053049; RRID: AB_2682025 Mouse anti-FIP200 (6B2) MFPL antibody facility N/A Rabbit anti-Stx17 Sigma Cat#HPA001204; RRID: AB_1080118 Mouse anti-LC3B (clone 2G6) nanoTools Cat#0260-100 Rabbit anti-GABARAP Cell Signaling Technology Cat#13733 Mouse anti-GFP Roche Cat#11814460001; RRID: AB_390913 Mouse anti-myc (clone 4A6) Millipore Cat#05-724; RRID: AB_310809 Rabbit anti-Atg19 Eurogentec N/A Rabbit anti-Ape1 Gift from Kraft lab (Institute for Biochem.

Techniques: Western Blot, Immunoprecipitation