rabbit polyclonal anti-hrd1  (Novus Biologicals)

Journal: bioRxiv

Article Title: GRASP55 Regulates Sorting and Maturation of the Lysosomal Enzyme β-Hexosaminidase A

doi: 10.1101/2024.10.16.618769

Figure Lengend Snippet: Depletion of GRASP55 results in decreased expression of the mature GNPTAB enzyme. A) Cell lysates from WT and 55KO GNPTAB-HA Knock-In HEK293T cells were probed with an HA antibody. In the absence of GRASP55, the mature form of GNPTAB-HA was reduced, while the pro-form was increased. Hrd1 was used as a loading control. B-D) Scatter plot summarizing quantification of pro-form and mature GNPTAB-HA from three independent blots (data presented as means ± S.D.). Statistical analysis was performed using Student’s t-test. *, p < 0.05; **, p < 0.01. E) Western blots of lysates from 55KO and 55R GNPTAB-HA Knock-In HEK cells using an HA antibody. In rescue cells, the mature form of GNPTAB expression increased, while the pro-form decreased compared to 55KO cells.

Article Snippet: The following antibodies were used: Mouse monoclonal anti-HEXA (Santa Cruz, Cat#376735), Rabbit polyclonal anti-GRASP55 (Proteintech Group, Cat# 10598-1-AP), Mouse monoclonal anti-beta-Actin (Proteintech Group, Cat# 66009-1-lg), Rabbit polyclonal anti-GAPDH (Proteintech Group, Cat#10494-1-AP), Rabbit polyclonal anti-Hexokinase1 (Proteintech Group, Cat#19662-1-AP), Mouse monoclonal anti-CD-MPR (DSHB, Cat#22d4), Rabbit polyclonal anti-CI-MPR (Proteintech Group, Cat#20253), Mouse monoclonal anti-GM130 (BD Biosciences, Cat# 610823), Mouse monoclonal anti-FLAG (Sigma, Cat#M1804), Mouse monoclonal anti-GFP (Proteintech Group Cat#66002-1-lg), Rabbit polyclonal anti-HRD1 (Proteintech Group Cat#13473-1-AP), Rabbit polyclonal anti-Lamp1 (Proteintech Group, CD107a), Rabit polyclonal anti-lamp2 (Proteintech Group, CD107b), Rabit polyclonal anti-GNPTAB (SAB2106865, Millipore Sigma).

Techniques: Expressing, Knock-In, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Intramembrane protease SPP defines a cholesterol-regulated abundance control of the mevalonate pathway enzyme squalene synthase

doi: 10.1016/j.jbc.2024.105644

Figure Lengend Snippet: SPP and TRC8 define a noncanonical ERAD pathway that acts on SQS in parallel to Hrd1 pathway. A , stability of endogenous SQS in Hek293T wt and Hek293TΔSPP cells assessed by cycloheximide (CHX) chase assay and analyzed by Western blot (WB) analysis. Equal protein loading was controlled by actin on a separate WB. Mean ± SD, n = 3 . B , SQS degradation assay in Hek293T wt, ΔTRC8, ΔMARCH6, and ΔTRC8ΔMARCH6 (ΔTΔM) double-deficient Hek293T cells. Mean ± SD, n = 3 . C , SQS degradation assay in ΔTΔM cells transfected with nontargeting (nt) or Hrd1 siRNA. Mean ± SD, n = 3 . See also Fig. S2 . ERAD, ER-associated degradation; SPP, signal peptide peptidase; SQS, squalene synthase.

Article Snippet: The following antibodies were used in this study: rabbit polyclonal anti-SPP (kind gift of C. Schaller), mouse monoclonal anti-FLAG (M2, Sigma-Aldrich, #F1804), rat monoclonal anti-HA (3F10, Sigma-Aldrich, #11867423001), mouse monoclonal anti-CLIMP63 (G1/296, Enzo Life Sciences, #ENZ-ABS669-0100), mouse monoclonal anti-β actin (AC-15, Sigma-Aldrich, #A1978), mouse monoclonal anti-ubiquitin (P4D1, Santa Cruz Biotechnology, #sc-8017), rabbit polyclonal anti-HO1 (Enzo Life Sciences, #ADI-SPA-896-F), rabbit polyclonal anti-SQLE (Proteintech, #12544-1-AP), rabbit monoclonal anti-SQS (Abcam, #ab109723), rabbit monoclonal anti-SQS (Abcam, #ab195046), mouse monoclonal anti-GFP (Sigma-Aldrich, #11814460001), mouse monoclonal anti-HMGCR (Merck, #MABS1233), rabbit polyclonal anti-Synoviolin (Hrd1) (Bethyl Laboratories, #A302-946A), rabbit polyclonal anti-Calnexin (Abcam,#ab22595), goat polyclonal anti-SREBP2 (Novus Biologicals, #AF7119), AlexaFluor488 goat anti-mouse IgG (H + L) (Invitrogen, #A-11029).

Techniques: Western Blot, Degradation Assay, Transfection

Journal: EMBO Reports

Article Title: DDX5 inhibits inflammation by modulating m6A levels of TLR2/4 transcripts during bacterial infection

doi: 10.1038/s44319-023-00047-9

Figure Lengend Snippet: ( A ) Redistribution of DDX5 after treatment of TLR2/TLR4 agonists. MEFs were treated with LPS, FSL-1, and Pam3CSK4 respectively, harvested to extract the nuclear and cytoplasmic protein for western blot assay to detect the expression of DDX5.β-Actin acted as a cytoplasmic reference control, and PCNA was a nuclear reference control. The expression of cytoplasmic DDX5 was quantified by the relative intensity of DDX5/β-Actin, and the expression of nuclear DDX5 was quantified by the relative intensity of DDX5/PCNA. The band intensity was measured by Image J software. ( B ) Interaction between Flag-Hrd1 and Myc-DDX5 in 293T cells detected by Co-IP. ( C ) DDX5 level in Hrd1 knockdown MEFs after treatment with LPS, FSL-1, and Pam3CSK4; NC served as the siRNA negative control. ( D ) DDX5 level in Flag-Hrd1- or Flag-Hrd1mutant (C329S)-expressing MEFs after treatment with LPS, FSL-1, and Pam3CSK4; Con served as the vector control. ( E ) Interaction between Hrd1 and DDX5 in MEFs after treatment with LPS, FSL-1, and Pam3CSK4 by Co-IP with an anti-DDX5 antibody. The interaction of Hrd1 and DDX5 was quantified by the band density of Hrd1/DDX5 in the IP system; the band density was measured by Image J software. ( F ) The co-localization of DDX5 and Hrd1 in mouse macrophages (Mø) after treatment of TLR2/TLR4 agonists. The mouse macrophages (Mø) were treated with TLR2/TLR4 agonists for 4 h, then fixed with 4% paraformaldehyde and stained with anti-DDX5 and anti-Hrd1before confocal microscopy. Right panels show the pixel intensity of red (Hrd1) and green (DDX5) from the black line in the merged image. Scale bars: 10 µm. ( G ) The K48 linked ubiquitin modification of DDX5 in Flag-Hrd1- or Flag-Hrd1 mutant (C329S)-expressing MEFs after treatment with LPS, FSL-1, and Pam3CSK4; Con served as the vector control. ( H ) The K48-linked ubiquitin modification of DDX5 in Hrd1 knockdown MEFs after treatment with LPS, FSL-1, and Pam3CSK4; NC served as the siRNA negative control. Data information: In ( A , E ), all data are represented as the mean ± SEM of three biologically independent samples. “ns” indicates no significant difference, * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t test). .

Article Snippet: Rabbit anti-Hrd1 polyclonal antibody , ABclonal Inc. , A2605.

Techniques: Western Blot, Expressing, Control, Software, Co-Immunoprecipitation Assay, Knockdown, Negative Control, Plasmid Preparation, Staining, Confocal Microscopy, Ubiquitin Proteomics, Modification, Mutagenesis

Journal: EMBO Reports

Article Title: DDX5 inhibits inflammation by modulating m6A levels of TLR2/4 transcripts during bacterial infection

doi: 10.1038/s44319-023-00047-9

Figure Lengend Snippet: In the absence of bacterial infection, DDX5/METTL3/METTL14 form a ternary complex. The complex promotes the m6A modification of TLR2/4 transcripts and affects the stability of transcripts, leading to their degradation via YTHDF2-dependent mRNAs decay, resulting in reduced expression of TLR2/4, which sequentially leads to the inactivation of the NF-κB-mediated inflammatory response and maintains homeostasis. Upon infection with pathogenic bacteria, DDX5 is degraded via the Hrd1-mediated ubiquitin-proteasome pathway, which prevents the formation of the mRNA methyltransferase ternary complex to specifically restrain m6A modification on TLR2/4 transcripts. TLR2/4 mRNAs are thus not degraded, but are excessively expressed, activating the NF-κB-mediated release of cytokines, such as IL-6, ultimately boosting the inflammatory response.

Article Snippet: Rabbit anti-Hrd1 polyclonal antibody , ABclonal Inc. , A2605.

Techniques: Infection, Modification, Expressing, Bacteria, Ubiquitin Proteomics

Journal: EMBO Reports

Article Title: DDX5 inhibits inflammation by modulating m6A levels of TLR2/4 transcripts during bacterial infection

doi: 10.1038/s44319-023-00047-9

Figure Lengend Snippet: Reagents and tools.

Article Snippet: Rabbit anti-Hrd1 polyclonal antibody , ABclonal Inc. , A2605.

Techniques: Recombinant, Luciferase, Plasmid Preparation, Sequencing, Transfection, Magnetic Beads, SYBR Green Assay, Protease Inhibitor, Software

Journal: Frontiers in Veterinary Science

Article Title: The H protein of attenuated canine distemper virus is degraded via endoplasmic reticulum-associated protein degradation

doi: 10.3389/fvets.2023.1214318

Figure Lengend Snippet: The degradation of CDV 851H protein via ERAD. (A) The inhibition of ERAD by Eevarestatin I increased the CDV 851H protein level. 293T cells were transfected with Flag-CDV H for 24 h, then were treated with Eevarestatin I (4 μM) for 4 h. The cell lysates were analyzed by Western blot using anti-Flag and anti-GAPDH antibodies. The gray values of protein bands were analyzed by ImageJ software and the ratio of target protein gray values to GAPDH was calculated. (B) Knockdown of Hrd1 increased the CDV 851H protein level. After transfecting 293T cells with siRNA for Hrd1 or si-NC for 24 h, the cells were transfected with H protein of CDV 851 and CDV NJ(11)2 for another 24 h. Western blot was used to detect the CDV H protein level and confirm the silencing efficacy of Hrd1-targeted siRNA. The gray values of protein bands were analyzed by ImageJ software and the ratio of target protein gray values to GAPDH was calculated. All results are presented as means ± SD obtained from at least three independent sample preparations. * p < 0.05, ** p < 0.01.

Article Snippet: The antibodies were obtained commercially: anti-Flag mouse monoclonal antibody (F1804, Sigma), anti-ATF6 rabbit polyclonal antibody (DF6009, Affinity), anti-Hrd1 rabbit polyclonal antibody (A2605, Abclonal), anti-HA mouse monoclonal antibody (BD-PM2095, Biodragon), anti-GAPDH mouse monoclonal antibody (60004-1-Ig, Proteintech), HRP-conjugated goat anti-mouse IgG (BF03001, Biodragon), and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (A0568, Beyotime).

Techniques: Inhibition, Transfection, Western Blot, Software, Knockdown