rabbit pab against gfp  (Proteintech)

Journal: bioRxiv

Article Title: Ubiquitin ligase CHFR impairs Tie2 signaling via K 48 -linked ubiquitylation and degradation of Akt1 in endothelial cells

doi: 10.64898/2026.03.31.715582

Figure Lengend Snippet: a-b CHFR deficiency in EC prevents LPS-induced ubiquitylation of Akt1. HLMVEC transfected with sc-siRNA or CHFR-siRNA. At 72 h post-transfection, cells were challenged with LPS (5 μg/ml) for 6 h in the presence of the proteasomal inhibitor MG132 (10 μM). Cell lysates were immunoprecipitated with anti-Akt1 mAb and blotted with antibodies specific to K 48 -linked poly-Ub or K 63 -linked poly-Ub ( n = 2 independent experiments) ( a ). Chfr fl/fl (WT) and Chfr ΔEC mice were challenged with LPS (10 mg/kg; i.p.) for 6 h. After the LPS challenge, lungs harvested were used to determine ubiquitylation of Akt1 as above in a (b) . c-f CHFR induces ubiquitylation of activated Akt1. c Control and CHFR-depleted HLMVEC were pretreated with inhibitors of PDK1 and mTORC2 for 30 min and then exposed to LPS (5 μg/ml). Thereafter, cell lysates were used for IB to assess phosphorylation of Akt1. d-e HLMVEC pretreated with or without PDK1 and mTORC2 inhibitors for 1 h and stimulated with LPS (6 h) in the presence of MG123 (10 μM) showed that CHFR binds and ubiquitylates phosphorylated Akt1. f HEK-293T cells were transfected with HA-tagged ubiquitin (HA-Ub) (0.5 μg/ml) alone or co-transfected with N-terminal GFP-tagged WT-CHFR (1.5 μg/ml), N-terminal pmCherry-tagged WT-Akt1 (1.5 μg/ml), and phosphorylation-defective Akt1 mutant (Akt1T308A/S473A) (1.5 μg/ml) plasmids. Thirty-six hours after transfection, cells were incubated with MG123 (10 μM) for 3h, and cell lysates were used to determine phosphorylation-dependent ubiquitylation of Akt1.

Article Snippet: Mouse mAb against β-actin (catalog #A5441; IB, 1:2000) was from Sigma-Aldrich Inc. Rabbit pAb against GFP (catalog #50430-2-AP; IB, 1:2000), rabbit pAb against CHFR (catalog #12169-1-AP; IB, 1:1000), rabbit pAb against Angiopoietin-2 (catalog #24613-1-AP; IB, 1:1000), rabbit pAb against β-catenin (catalog #61067-2-AP; IB, 1:1000), and rabbit pAb against mCherry (catalog #26765-1-AP; IB, 1:1000) were from Proteintech.

Techniques: Transfection, Immunoprecipitation, Control, Phospho-proteomics, Ubiquitin Proteomics, Mutagenesis, Incubation

Journal: bioRxiv

Article Title: Ubiquitin ligase CHFR impairs Tie2 signaling via K 48 -linked ubiquitylation and degradation of Akt1 in endothelial cells

doi: 10.64898/2026.03.31.715582

Figure Lengend Snippet: a Schematics of the domain structures of human CHFR WT and mutants lacking forkhead-associated domain (ΔFHA-CHFR), RING finger domain (ΔRF-CHFR), cysteine-rich domain (ΔCR-CHFR), or poly-ADP ribose binding zinc-finger domain (ΔPBZ-CHFR) used in experiments. b Immunoblot showing expression of eGFP-tagged CHFR (WT) and CHFR mutants (1.5 μg/ml), along with pmCherry-tagged WT-Akt1 (1.5 μg/ml) in HEK-293T cells. c Transfected HEK-293T cells were used for anti-GFP agarose beads pull-down assays. Results show that WT-CHFR and CHFR mutants bind to WT-Akt1 in vitro . Bottom panel shows quantification of CHFR binding to Akt1 as a ratio of Akt1-to-GFP-CHFR. arb. units, arbitrary units. d HEK-293T cells transfected with WT-HA-Ub (0.5 μg/ml) alone or co-transfected with WT-CHFR (1.5 μg/ml), ΔFHA-CHFR (1.5 μg/ml), ΔRF-CHFR (1.5 μg/ml), and WT-Akt1 (1.5 μg/ml) were used to assess ubiquitylation of Akt1. At 36 h after transfection, cells were incubated with MG132 (10 μM) for 3 h, and then cell lysates were used for IB analysis. e-f HEK-293T cells transfected with WT-HA-Ub (0.5 μg/ml) or HA-tagged Ubiquitin where all Lysin (K) residues were mutated to Alanine (A) except at K48 or K63, along with WT-CHFR, and WT-Akt1 were used to determine CHFR-mediated linkage specific polyubiquitylation of Akt1.

Article Snippet: Mouse mAb against β-actin (catalog #A5441; IB, 1:2000) was from Sigma-Aldrich Inc. Rabbit pAb against GFP (catalog #50430-2-AP; IB, 1:2000), rabbit pAb against CHFR (catalog #12169-1-AP; IB, 1:1000), rabbit pAb against Angiopoietin-2 (catalog #24613-1-AP; IB, 1:1000), rabbit pAb against β-catenin (catalog #61067-2-AP; IB, 1:1000), and rabbit pAb against mCherry (catalog #26765-1-AP; IB, 1:1000) were from Proteintech.

Techniques: Binding Assay, Western Blot, Expressing, Transfection, In Vitro, Incubation, Ubiquitin Proteomics

Journal: Cell reports

Article Title: VezA/vezatin facilitates proper assembly of the dynactin complex in vivo

doi: 10.1016/j.celrep.2024.114943

Figure Lengend Snippet: (A) A western blot of total extracts from wild-type ( vezA +) and Δ vezA strains containing p150-GFP, p50-GFP, or Arp11-GFP probed with an anti-GFP antibody. Ponceau S straining of the same blot is also shown. Note that p50 with a predicted molecular weight of ~50 kDa runs at a position higher than expected, as p50-GFP and Arp11-GFP (Arp11 is predicted to be 62 kDa) are at about the same position on the SDS gel. (B) Western blots of total extracts showing p150-GFP and Arp11 in the same lanes. Please see for uncropped blots. (C) Western blots of total extracts showing p50-GFP and Arp11 in the same lanes. Please see for uncropped blots. (D) The intensity ratios of p150-GFP or p50-GFP to Arp11 in wild type ( vezA +) and the Δ vezA mutant. The ratios in vezA + samples are set as 1. Scatterplots with mean and SD values as well as the p value were generated by Prism 10 (Student’s t test, two tailed, unpaired). The values were generated from three western blot experiments ( n = 3 for all). (E) Western blots of total extracts showing p150, p50-S, and Arp11-GFP signals in the same lanes. Please see for uncropped blots. (F) The ratios of the intensity value of Δ vezA to that of wild-type vezA (Δ vezA / vezA +) are shown for both p150 and p50-S, and these ratios are relative to the ratio for Arp11-GFP, which was set as 1. Scatterplots with mean and SD values as well as p values were generated by Prism 10 (ordinary one-way ANOVA with Dunnett’s multiple comparisons test). The values were generated from three western blot experiments ( n = 3 for all). (G) Western blots showing that the amounts of p150, p50-S, and Arp1 pulled down with Arp11-GFP are lowered in the Δ vezA mutant. Please see for uncropped blots. Please also see for uncropped blots including a negative control for the pull-down (a strain containing p50-S but no GFP) as well as a negative control for total extract western analysis (a strain with no p50-S). (H) A quantitative analysis of the effect of Δ vezA on the amounts of p150, p50-S, and Arp1 pulled down with Arp11-GFP. The ratios of the intensity value of Δ vezA to that of wild-type vezA (Δ vezA / vezA +) are shown for each component, and these ratios are relative to the ratio for Arp11-GFP, which was set as 1. Scatterplots with mean and SD values as well as p values were generated by Prism 10 (ordinary one-way ANOVA with Dunnett’s multiple comparisons test). The values were generated from three independent pull-down experiments ( n = 3 for all). (I) A quantitative analysis of the effect of Δ vezA on the amounts of proteins pulled down with Arp11-GFP. The ratios of the total peptide number of Δ vezA to that of wild-type vezA (Δ vezA / vezA +) for each protein are shown, and these ratios are relative to the ratio for Arp11-GFP, which was set as 1. Scatterplots with mean and SD values as well as p values were generated by Prism 10 (ordinary one-way ANOVA with Dunnett’s multiple comparisons test). The values were generated from three mass spectrometry analyses of three independent pull-down experiments ( n = 3 for all).

Article Snippet: The rabbit polyclonal antibody against GFP was from Takara Bio Inc. (Catalog number: 632592).

Techniques: Western Blot, Molecular Weight, SDS-Gel, Mutagenesis, Generated, Two Tailed Test, Negative Control, Mass Spectrometry

Journal: Cell reports

Article Title: VezA/vezatin facilitates proper assembly of the dynactin complex in vivo

doi: 10.1016/j.celrep.2024.114943

Figure Lengend Snippet:

Article Snippet: The rabbit polyclonal antibody against GFP was from Takara Bio Inc. (Catalog number: 632592).

Techniques: Produced, Virus, Recombinant, Protease Inhibitor, Isolation, Gel Extraction, Software, Mass Spectrometry

Journal: PLoS ONE

Article Title: Rab7 Mutants Associated with Charcot-Marie-Tooth Disease Exhibit Enhanced NGF-Stimulated Signaling

doi: 10.1371/journal.pone.0015351

Figure Lengend Snippet: (A) BHK cells were transfected with GFP-tagged Rab7 wild-type and CMT2B disease mutants. After 16 h of transfection, cells were fixed and permeabilized by saponin treatment. Enlarged perinuclear vesicular staining was observed for the CMT2B disease mutants. (B) PC12 cells were stimulated with NGF (200 ng/ml) for 15–60 min as indicated. Cells were lysed and samples of cell lysates were immunoblotted for TrkA and Rab7. Lysate samples containing equal amounts of protein were immunoprecipitated (IP) with a pAb directed against Rab7 and immunoblotted for TrkA. Subsequently, the membrane was stripped and reprobed for Rab7. (C) PC12 cells were transfected with wild-type GFP-Rab7 and stimulated with NGF for 60 min. Samples containing equal amounts of protein were immunoprecipitated with antibodies against GFP to isolate the GFP-Rab7 and subsequently immunoblotted for TrkA. The membrane was stripped and reprobed with antibodies against GFP. (D) PC12 cells were transfected with GFP-tagged Rab7 CMT2B disease mutants and stimulated with NGF for 60 min. Equal amounts of lysate samples were immunoprecipitated with antibodies against GFP to precipitate the GFP-Rab7 and subsequently immunoblotted for TrkA. The membrane was stripped and reprobed with antibodies against GFP. (B–D) Representative blots from one of three independent experiments are shown in each case.

Article Snippet: The supernatant (precleared lysate) was transferred in a clean microfuge tube and incubated with 3 µg rabbit pAb directed against GFP (Invitrogen) for 2 h at 4°C.

Techniques: Transfection, Staining, Immunoprecipitation