Journal: Cell systems

Article Title: Comparative analysis of immune cells reveals a conserved regulatory lexicon

doi: 10.1016/j.cels.2018.01.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Covaris tru-ChIP Chromatin Shearing and Reagent Kit , Covaris , 520154.

Techniques: Software

Journal: eLife

Article Title: CDK1 controls CHMP7-dependent nuclear envelope reformation

doi: 10.7554/eLife.59999

Figure Lengend Snippet: ( A ) Cartoon depiction of C-terminal fragments of CHMP7 used to analyse NESs. Colouring matches that in . MIMC2A involves replacement of Helix6 with the MIM from CHMP2A; ES-RR involves charge reversal of E424R, S429R to restore the charge distribution of a type-1 MIM. ( B ) Glutathione-sepharose captured fractions and lysates from 293 T cells transiently expressing YFP-VPS4 MIT and either GST, GST-CHMP7 393-CT, GST-CHMP7 393-CT+MIM, or GST-CHMP7 393-CT ES-RR were examined by western blotting with antisera raised against GST or GFP (N = 3) and quantified by infrared imaging (N = 2). Mean ± S.D. presented, p-values generated with one-way ANOVA with Tukey’s multiple comparison. CHMP7 393-CT δH6 + MIM C2A restored binding to YFP-VPS4 MIT (p = 0.038). CHMP7 393-CT ES-RR (E424R, S429R) was created to restore the Type-1 MIM and permitted binding to YFP-VPS4 MIT (p = 0.003). ( C) . Helical wheel displaying overlapping sequences of NES2 and the putative type-1 MIM within CHMP7 Helix6. Figure 2—figure supplement 2—source data 1. CHMP7's C-terminus does not bind to VPS4's MIT domain.

Article Snippet: An antibody against GAPDH (MAB374) was from Millipore; Calnexin (ab22595) was from Abcam; Tubulin (DM1A) was from Sigma; CHMP2A (10477–1-AP) was from Proteintech; IST1 (51002–1-AP) was from Proteintech; CHMP7 (16424–1-AP) was from Proteintech; GFP (7.1/13.1) was from Roche; mCherry (ab167453) was from Abcam; HA.11 (16B12) was from (Biolegend); γH2AX (05–636) was from Sigma; 53BP1 (NB100-305) was from (Novus Biologicals).

Techniques: Expressing, Western Blot, Imaging, Generated, Comparison, Binding Assay

Journal: eLife

Article Title: CDK1 controls CHMP7-dependent nuclear envelope reformation

doi: 10.7554/eLife.59999

Figure Lengend Snippet:

Article Snippet: An antibody against GAPDH (MAB374) was from Millipore; Calnexin (ab22595) was from Abcam; Tubulin (DM1A) was from Sigma; CHMP2A (10477–1-AP) was from Proteintech; IST1 (51002–1-AP) was from Proteintech; CHMP7 (16424–1-AP) was from Proteintech; GFP (7.1/13.1) was from Roche; mCherry (ab167453) was from Abcam; HA.11 (16B12) was from (Biolegend); γH2AX (05–636) was from Sigma; 53BP1 (NB100-305) was from (Novus Biologicals).

Techniques: Cloning, Transfection, Construct, Retroviral, Plasmid Preparation, Expressing, Modification, Sequencing, Produced, Recombinant, Protein Purification, Control

Journal: The Journal of Cell Biology

Article Title: ALIX- and ESCRT-III–dependent sorting of tetraspanins to exosomes

doi: 10.1083/jcb.201904113

Figure Lengend Snippet: ALIXΔPRR stimulates the release of exosomes containing tetraspanins. (A and B) HeLa GFP-CHMP4B cells transfected for 18 h with ALIXΔPRR (ΔPRR) or GFP as a control and incubated with exosome-free medium for 24 h. The cell medium was collected, and exosomes were isolated by differential centrifugation. The cell lysate and the exosome samples were analyzed by Western blotting using antibodies against the indicated proteins. The same amount of protein was loaded for each condition (A). The amounts of protein were quantified by densitometry and are expressed relative to free GFP. In B, boxes, mean; error bars, ±SD ( n = 3, from three independent experiments); t test; *, P < 0.05, ***, P < 0.001; ns, not significant. (C and D) HeLa GFP-CHMP4B cells were treated with siRNA against ALIX or nontarget siRNAs (siCtrl) and incubated with exosome-free medium for 24 h. Exosomes were isolated as in A and B, and then cell lysates and exosomes (C) were analyzed by Western blotting as in A and B. The relative amount of protein in exosome fractions was quantified by densitometry (D). Boxes, mean; error bars, ±SD ( n = 3, from three independent experiments); t test; *, P < 0.05, ***, P < 0.001; ns, not significant. (E and F) The experiment was as in C and D except that siRNAs against CHMP6 and not against ALIX were used. The upper band in the syntenin blot of siCtrl is CHMP6, because the same membrane was reused for incubation with both antibodies. The relative amounts of protein were then quantified by densitometry (F). Boxes, mean; error bars, ±SD ( n = 3, from three independent experiments); t test; *, P < 0.05, **, P < 0.01. (G and H) HeLa GFP-CHMP4B cells were transfected with WT CD9 (WT) tagged with V5 or with the V5-CD9/3R mutant (3R). Total cell lysates and exosomes prepared as in A and B were analyzed by Western blotting using antibodies against V5, ALIX, flotillin-1, and syntenin (G). The V5 blot is shown after short and long exposure, so that WT V5-CD9 and V5-CD9/3R can be better compared. The relative amounts of CD9 were quantified by densitometry (H). Boxes, mean; error bars, ±SD ( n = 3, from three independent experiments); t test; *, P < 0.05.

Article Snippet: The antibodies against STAM1 (12434-1-AP), HD-PTP (10472-1-AP), TSG101 (14497-1-AP), CHMP6 (16278-1-AP), VPS4B (17673-1-AP), CHMP1A (15761-1-AP), and CHMP3 (15472-1-AP) were from Proteintech; against FLAG (F3165) and tubulin (T9026) from Sigma-Aldrich; against CHMP4A (H-52) and VPS22 (EAP30 C-11) from Santa Cruz Biotechnology; against CHMP4B (ab105767), BROX (ab193008), flotillin-1 (ab41927), CD9 (ab2215), CD81 (ab79559), CD63 (ab59479), syntenin (ab19903), calnexin (ab13504), LAMP1 (ab25630), integrin α6 (ab181551), and integrin β3 (ab119992) from Abcam; against HRS (GTX101718) from Genetex; against EEA1 (ALX-210-239-C100) and conjugated ubiquitin (FK2) from Enzo Life Sciences; against LAMP1 (D2D11) from Cell Signaling Technology; against GFP (11814460001) from Roche; against V5 (R960-25) from Thermo Fisher Scientific; against EGFR (20-ES04, for Western blot) from Fitzgerald; against EGFR (555996, for immunofluorescence), cathepsin D (610801), and GM130 (610822) from BD Biosciences; and against ALIX (pab0204) from Covalab.

Techniques: Transfection, Control, Incubation, Isolation, Centrifugation, Western Blot, Membrane, Mutagenesis