pan ubiquitin antibody  (Bio-Rad)

Journal: The Journal of Cell Biology

Article Title: Rsp5/NEDD4 and ESCRT regulate TDP-43 toxicity and turnover via an endolysosomal clearance mechanism

doi: 10.1083/jcb.202212064

Figure Lengend Snippet: TDP-43 toxicity, aggregation, and toxicity depend on the E3 ubiquitin ligase Rsp5. (A) Representative serial dilution growth assay of WT and rsp5-3 strains transformed with the empty vector (pRB43) or untagged TDP-43 plasmid (pRB232) grown at 25°C or 30°C as indicated. N = 3. (B) WT and rsp5-3 strains were transformed with TDP-43-mRuby2 plasmid and grown to mid-logarithmic growth phase at 25°C, and samples were imaged. Cultures were then incubated at 35°C for 16 h and imaged again. **, P < 0.01 by one way analysis of variance with Tukey’s post hoc test; N = 3. Scale bar, 5 µm. (C) WT and rsp5-3 strains were transformed with TDP-43-mRuby2 plasmid and grown to mid-logarithmic growth phase at 25°C before being treated with 0.2 mg/ml cycloheximide for 0, 6, 12, and 24 h. At time 0, cultures were either shifted to 35°C or kept at 25°C for the remainder of the time course. TDP-43-mRuby2 levels were detected by western blot using anti–TDP-43 antibody and normalized to GAPDH loading control. **, P < 0.01; ***, P < 0.001 by one-way analysis of variance with Tukey’s post hoc test; N = 3. (D) Strains endogenously expressing GFP-Rsp5, GFP-Rim8, GFP-Ssh4, and GFP-Ear1 were transformed with a TDP-43-mRuby2 plasmid (pRB194) and imaged at mid-log phase growth. Percentage values indicate colocalization frequency of TDP-43-mRuby2 foci with GFP foci. N = 3; scale bar, 5 µm. (E and F) Representative serial dilution growth assay of WT strain transformed with an empty vector control (pRB43) or untagged TDP-43 plasmid (pRB232) and with the empty vector control (pRB511) or Rsp5 overexpression plasmid (pRB476). N = 3 (F) WT, vps4Δ , and vps28Δ strains were transformed with TDP-43-YFP (pRB109) and also transformed with Rsp5 overexpression (pRB476) or empty vector control plasmid (pRB511) and grown to mid-logarithmic growth phase. TDP-43-YFP (pRB109) was detected by western blot using anti–TDP-43 antibody. TDP-43 levels were normalized to GAPDH loading control. **, P < 0.01 by Welch’s two-tailed T test; N = 3. Error bars in all graphical panels represent standard error of the mean. Source data are available for this figure: .

Article Snippet: Primary antibodies were as follows: anti-GFP ( QAB10298 ; enQuireBio, dilution 1:5,000), anti-GAPDH (MA5-15738; Invitrogen, dilution 1:5,000), anti-αTubulin (66031; Proteintech, dilution 1:5,000), anti-TDP-43 (10782-2-AP; Proteintech, dilution 1:5,000), anti-NEDD4 (21698-1-AP; Proteintech, dilution 1:1,000), anti-pan Ubiquitin (sc-8017; Santa Cruz, dilution 1:1,000), K63 Ubiquitin (ab179434; Abcam, dilution 1:500), anti-K48 Ubiquitin (D905; Cell Signaling Technology, dilution 1:500), and anti-mCherry (ab167453; Abcam, dilution 1:1,000).

Techniques: Ubiquitin Proteomics, Serial Dilution, Growth Assay, Transformation Assay, Plasmid Preparation, Incubation, Western Blot, Control, Expressing, Over Expression, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Rsp5/NEDD4 and ESCRT regulate TDP-43 toxicity and turnover via an endolysosomal clearance mechanism

doi: 10.1083/jcb.202212064

Figure Lengend Snippet: HEK293A cells were transfected with HA-ubiquitin (pRB506) or both HA-Ub and NEDD4-mCherry (pRB334) and grown for 2 days before TDP-43 was immunoprecipitated. (A) Representative biological replicate of K63 and K48 Ub signal, NEDD4, TDP-43, and α-tubulin from a single biological replicate; run on a 7% SDS-PAGE gel. (B) As in A, except Pan-Ub signal is shown in place of K63 and K48 on a gradient gel. Arrowheads indicate full-length species of TDP-43 and NEDD4-mCherry, with presumed degradation products/protein isoforms shown below. Trends shown were reproduced across five biological replicates. Source data are available for this figure: .

Article Snippet: Primary antibodies were as follows: anti-GFP ( QAB10298 ; enQuireBio, dilution 1:5,000), anti-GAPDH (MA5-15738; Invitrogen, dilution 1:5,000), anti-αTubulin (66031; Proteintech, dilution 1:5,000), anti-TDP-43 (10782-2-AP; Proteintech, dilution 1:5,000), anti-NEDD4 (21698-1-AP; Proteintech, dilution 1:1,000), anti-pan Ubiquitin (sc-8017; Santa Cruz, dilution 1:1,000), K63 Ubiquitin (ab179434; Abcam, dilution 1:500), anti-K48 Ubiquitin (D905; Cell Signaling Technology, dilution 1:500), and anti-mCherry (ab167453; Abcam, dilution 1:1,000).

Techniques: Transfection, Ubiquitin Proteomics, Immunoprecipitation, SDS Page

Journal: Theranostics

Article Title: H3K18 lactylation-mediated SPHK1-SIRT1 feedback loop accelerates pyroptosis of tubular epithelial cells in sepsis-associated acute kidney injury

doi: 10.7150/thno.122991

Figure Lengend Snippet: SPHK1 disrupts mitochondrial homeostasis by inducing ubiquitin-dependent SIRT1 degradation via phosphorylation at Ser47. (A) Protein levels of SIRT1, PGC-1α acetylation, PGC-1α and TFAM in HK-2LPS cells transfected with siNC or siSPHK1, with or without lactate treatment (n = 5). (B) CHX chase assay demonstrating SIRT1 protein degradation kinetics in lactated-treated HK-2LPS cells transfected with siNC or siSPHK1. (C) SPHK1-induced SIRT1 degradation depends on the ubiquitin-proteasome pathway in lactated-treated HK-2LPS cells. (D) Ubiquitination of SIRT1 in lactated-treated HK-2LPS cells. (E and F) Co-IP detection showed SPHK1-SIRT1 interaction in lactated-treated HK-2LPS cells. (G) Protein levels of SIRT1 phosphorylated at Ser47 and SIRT1 in HK-2LPS cells transfected with siNC or siSPHK1, with or without lactate treatment (n = 5). (H and I) Protein levels of SIRT1 ubiquitination, SIRT1 phosphorylated at Ser47, SIRT1, PGC-1α acetylation, PGC-1α and TFAM and (J) representative immunofluorescence images of mtDNA leakage (Scale bars = 10 μm, up; 2.5 μm, down) in lactated-treated HK-2LPS cells co-transfected with siNC or siSPHK1 and wild-type SIRT1 (SIRT1-WT) or mutant SIRT1 (SIRT1-S47A) (n = 5).

Article Snippet: After blocking the membranes with TBST buffer containing 5% skim milk or 5% BSA, the membranes were incubated at 4 °C overnight with primary antibodies against SPHK1 (BA2865, Boster), KIM-1 (NBP1-76701, Novus), GSDMD (39754, CST), Caspase-1 (83383, CST), H3K18la (PTM-1427RM, PTM Bio), Cleaved Caspase-1 (89332, CST), P300 (ab275378, Abcam), Cleaved Caspase-1 (4199, CST), NLRP3 (P60622R3, Abmart), SIRT1 (13161-1-AP, Proteintech), NLRC4 (PB0658, Boster), PGC-1α (66369-1-Ig, Proteintech), Acetylated-Lysine (9441, CST), p-SIRT1 (SAB4301426, MilliporeSigma), Pan-Kla (PTM-1401RM, PTM Bio), Pan-Ubiquitin (PTM-1124RM, PTM Bio), AIM2 (20590-1-AP, Proteintech), NLRP1 (12256-1-AP, Proteintech), Histone H3 (PTM-1002RM, PTM Bio) and β-actin (AC026, Abclonal).

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Transfection, Co-Immunoprecipitation Assay, Immunofluorescence, Mutagenesis