Journal: Experimental and Therapeutic Medicine

Article Title: Clinical characteristics and prognostic analysis of patients with HIV and glioma: A case series and literature review

doi: 10.3892/etm.2024.12380

Figure Lengend Snippet: Characteristics of HIV-positive patients with brain glial tumors treated at Beijing Ditan Hospital.

Article Snippet: The primary antibodies CD31 (cat. no. ZA-0568), CD68 (cat. no. ZM-0060), CD163 (cat. no. ZM-0428), IDH1 R132H mutation-specific (cat. no. ZM-0447) and Ki-67 (cat. no. ZM-0166) were purchased from ZSGB-BIO (OriGene Technologies, Inc.) and used at a dilution of 1:100.

Techniques:

Journal: eLife

Article Title: A nuclear-based quality control pathway for non-imported mitochondrial proteins

doi: 10.7554/eLife.61230

Figure Lengend Snippet: ( A ) 35 S-labeled Su9-DHFR was imported into mitochondria isolated from yeast overexpressing the indicated Ilv2-GFP variants. ( B ) Western blot showing protein levels in mitochondria isolated from yeast overexpressing the indicated Ilv2-GFP variants. ( C ) 35 S-labeled Su9-DHFR was imported into mitochondria isolated from yeast expressing endogenous Ilv2-GFP ± FCCP. ( D ) Western blot showing protein levels in mitochondria isolated from yeast expressing endogenous Ilv2-GFP ± FCCP. ( E ) Yeast expressing Idh1-GFP and Tom70-mCherry in WT or Ilv2 (untagged) overexpressing strains ± FCCP. Bars = 2 μm. ( F ) Quantification of ( E ). ( G ) 35 S-labeled Su9-DHFR was imported into mitochondria isolated from yeast expressing endogenous Ilv2-GFP or Ilv2-NES-GFP ± FCCP. ( H ) Western blot showing protein levels in mitochondria isolated from yeast expressing endogenous Ilv2-GFP or Ilv2-NES-GFP. For ( A ), ( C ), and ( G ), a mixture of antimycin A, oligomycin, and valinomycin (AVO) was used to dissipate the membrane potential. Non-imported proteins were proteolytically removed with proteinase K and the import was analyzed by SDS–PAGE and autoradiography. P = precursor and M = mature.

Article Snippet: Membranes were blocked and probed in blocking buffer (1× PBS, 0.05% Tween 20, 5% non-fat dry milk) using the primary antibodies for GFP (1814460001, Sigma Millipore), HA (11583816001, Sigma Millipore), Ilv2 (gift from Dr. Agnieszka Chacinska, International Institute of Molecular and Cell Biology), Tom70, Tom20, Tim54, Tim50, Tim18 (gifts from Dr. Nikolaus Pfanner, University of Freiburg), OM45 (gift from Dr. Carla Koehlar, UCLA), Idh1 (Ap31099PU-N, ORIGENE) or Pgk1 (22C5D8, Abcam), and HRP-conjugated secondary antibodies (715-035-150, Jackson Immunoresearch).

Techniques: Labeling, Isolation, Western Blot, Expressing, Membrane, SDS Page, Autoradiography

Journal: eLife

Article Title: A nuclear-based quality control pathway for non-imported mitochondrial proteins

doi: 10.7554/eLife.61230

Figure Lengend Snippet:

Article Snippet: Membranes were blocked and probed in blocking buffer (1× PBS, 0.05% Tween 20, 5% non-fat dry milk) using the primary antibodies for GFP (1814460001, Sigma Millipore), HA (11583816001, Sigma Millipore), Ilv2 (gift from Dr. Agnieszka Chacinska, International Institute of Molecular and Cell Biology), Tom70, Tom20, Tim54, Tim50, Tim18 (gifts from Dr. Nikolaus Pfanner, University of Freiburg), OM45 (gift from Dr. Carla Koehlar, UCLA), Idh1 (Ap31099PU-N, ORIGENE) or Pgk1 (22C5D8, Abcam), and HRP-conjugated secondary antibodies (715-035-150, Jackson Immunoresearch).

Techniques: Recombinant, Plasmid Preparation, Generated, Protease Inhibitor, Staining, Cloning, Software

Journal: The Journal of Biological Chemistry

Article Title: Antibody validation for Western blot: By the user, for the user

doi: 10.1074/jbc.RA119.010472

Figure Lengend Snippet: Validation of IDH1 antibody using purified recombinant protein in multicolor and chemiluminescent Western blotting. Multicolor and chemiluminescent Western blottings were performed using 10% Bis-Tris SDS-polyacrylamide gel and MOPS buffer system to validate the IDH1 antibody using a purified recombinant IDH1 protein (0.16 μg) containing a c-Myc tag in addition to HEK293T and HeLa whole-cell lysates. A, c-Myc protein tag present on the purified IDH1 recombinant protein is detected in the 700-nm channel ( red ) at 50 kDa via mouse anti-c-Myc antibody (ab32;1 μg/ml) using IRDye 680RD goat anti-mouse IgG (H + L) for detection. Some overspill of the recombinant protein into neighboring lanes is observed ( white box ). B, IDH1 recombinant protein and endogenous IDH1 protein, present in HEK293T and HeLa, is detected in the 800-nm channel ( green ) at 55 and 50 kDa, respectively, using rabbit anti-IDH1 antibody (ab172964; 1.2 μg/ml) and IRDye 800CW goat anti-mouse IgG (H + L) for detection. C, when both 700- and 800-nm channels are displayed, the signal from ab32 and ab172964 overlaps at 50 kDa, identifying the c-Myc–tagged IDH1 protein. No overlap is seen for the endogenous IDH1 present in HEK293T and HeLa whole-cell lysates. A–C , lysates loaded per lane are as follows: 20 μg of blocking buffer: Odyssey blocking buffer (TBS); imager: Odyssey® CLx; resolution: 169 μm; intensity: auto mode. Chameleon TM Duo pre-stained protein ladder for accurate sizing of protein bands. D, single blot was split into two halves ( green line ) to be incubated with either rabbit anti-IDH1 antibody (ab172964; 0.115 μg/ml) or the corresponding rabbit monoclonal IgG isotype control (ab172730; 0.166 μg/ml) to detect the endogenous IDH1 protein present in HeLa and HEK293T as well IDH1 recombinant protein. Both halves were incubated with HRP-conjugated goat anti-mouse IgG (H + L). E, single blot was split into two halves ( green line ) to be incubated with either mouse anti-c-Myc antibody (ab32; 1 μg/ml) or the corresponding mouse monoclonal IgG1 isotype control (ab18443; 1 μg/ml) to detect c-Myc protein tag present on the purified IDH1 recombinant protein but absent in HEK293T and HeLa whole-cell lysates. Both halves were incubated with HRP-conjugated goat anti-rabbit IgG (H + L). Blots were detected with WesternSure® PREMIUM chemiluminescent substrate (LI-COR 926–95000) and imaged on an Odyssey® Fc with the following resolution: 125 μm and exposure of 2 min. Lysate loaded per lane: 20 μg; protein ladder: WesternSure® pre-stained chemiluminescent protein ladder (LI-COR 926-980000); blocking buffer: intercept blocking buffer (TBS); intercept T20 (TBS) antibody diluent.

Article Snippet: Isocitrate dehydrogenase (IDH1) (NM_005896) human recombinant protein (OriGene no. TP310582), IDH1 (NM_005896) human overexpression lysate supplied with parental HEK293T lysate (OriGene no. LY401782; HEK293T LY500001; lot no. 0076CF), and IDH1 knockout cell lysate (supplied with parental HeLa control lysate) (Origene no. LC810112, LC810Hela; lot no. 1601) were mixed with either 2× protein loading buffer (PLB) (LICOR no. 928-40004) or 2× SDS buffer (OriGene) and denatured by boiling at 97 °C for 5 min.

Techniques: Biomarker Discovery, Purification, Recombinant, Western Blot, Blocking Assay, Staining, Incubation, Control

Journal: Ecotoxicology and environmental safety

Article Title: Chronic nickel (II) exposure induces the stemness properties of cancer cells through repressing isocitrate dehydrogenase (IDH1).

doi: 10.1016/j.ecoenv.2021.112031

Figure Lengend Snippet: Fig. 4. Ni(II) exposure mediated the cancer stem-like properties by regulating IDH1 expression. (A) IDH1 expression was measured by western blotting in cells after culture for 24 h. (B) A549 and RKO cells were transfected with shRNA against IDH1, and the cells were collected to measure the sphere-formation rate. (C) The effect of IDH1 overexpression on the cell sphere number was measured in the A549P40 and RKOP40 cells. (D) SOX2 expression was measured by western blotting in cancer cells stably transfected with IDH. (E) NAPDH synthesis was analyzed in the nickel-treated cells and compared with the corresponding control cells after culture for 24 h. Notes: The data are shown as the mean ± SD from three independent experiments. Right column bar, quantification of the protein expression (4A and 4D) or quantification of the sphere number (4B and 4C). A549P40: A549 cells exposed to 100 µM Ni(II) for 40 passages. RKOP40: RKO cells exposed to 100 µM Ni(II) for 40 passages. A549Con: A549 cells exposed to 0 µM Ni(II) for 40 passages. RKO Con: RKO cells exposed to 0 µM Ni(II) for 40 passages.

Article Snippet: The primary antibodies against IDH1 (Cell Signaling Technology, #3997, 1:1000), HIF-1ɑ (Cell Signaling Technology, #36169, 1:1000), SOX2 (Cell Signaling Technology, #3579, 1:500) and β-actin (Cell Signaling Technology, #8457, 1:1500) and the secondary antibody (Cell Signaling Technology, #7074/7056 1:5000) were incubated for 1 h each.

Techniques: Expressing, Western Blot, Transfection, shRNA, Over Expression, Stable Transfection, Control

Journal: Ecotoxicology and environmental safety

Article Title: Chronic nickel (II) exposure induces the stemness properties of cancer cells through repressing isocitrate dehydrogenase (IDH1).

doi: 10.1016/j.ecoenv.2021.112031

Figure Lengend Snippet: Fig. 6. IDH1 negatively correlates with poor survival in READ. (A) IDH1 expression downregulation in stages II to IV in rectum adenocarcinoma (READ) according to GEPIA analysis. (B) Kaplan-Meier estimates of overall survival (OS) of IDH1 in READ. (C) Kaplan-Meier estimates of relapse-free survival (RFS) of IDH1 in READ.

Article Snippet: The primary antibodies against IDH1 (Cell Signaling Technology, #3997, 1:1000), HIF-1ɑ (Cell Signaling Technology, #36169, 1:1000), SOX2 (Cell Signaling Technology, #3579, 1:500) and β-actin (Cell Signaling Technology, #8457, 1:1500) and the secondary antibody (Cell Signaling Technology, #7074/7056 1:5000) were incubated for 1 h each.

Techniques: Expressing

Journal: ACS nano

Article Title: Copper Oxide Nanoparticles Impair Mouse Preimplantation Embryonic Development through Disruption of Mitophagy-Mediated Metabolism.

doi: 10.1021/acsnano.4c09734

Figure Lengend Snippet: Figure 6. CuONPs impair TCA cycle and reduce α-KG production. (A) Schematic diagram of the TCA cycle. Cs, Citrate Synthase; Aco, aconitase; Idh, isocitrate dehydrogenase. (B) Heatmap displaying the expression levels of TCA cycle-related genes in control and CuONPs- treated blastocyst from our RNA-Seq data. (C) Immunofluorescence images (left) and statistical analysis (right) of Aco2 in control and CuONPs-treated (n = 15 in each group) blastocyst. Scale bar: 20 μm. (D) Immunofluorescence images (left) and statistical analysis (right) of Idh1 in control (n = 14) and CuONPs-treated (n = 15) blastocyst. Scale bar: 20 μm. (E) Western blotting showing the protein levels of Aco2 and Idh1 in control and CuONPs-treated blastocyst. (F) Heatmap displaying the relative abundance of metabolites in control and CuONPs-treated blastocyst. For each group, n = 5 biological replicates. Each biological replicate collected 120 blastocysts. (G) The concentration of α-KG in blastocysts (left) and mESCs (right) (n = 120 in each group). (H) Representative images (left) and blastocyst rate (right) in control (n = 32), CuONPs (n = 54), and CuONPs+α-KG (n = 52) groups. Scale bar: 100 μm. (I) Representative images (left) and live birth rate (right) after blastocyst transplantation in CuONPs and CuONPs+α-KG (n = 12 in each group) groups. (J) Representative images of teratoma in CuONPs and CuONPs+α-KG groups. (K) H&E staining of teratoma showing three germ layers in CuONPs and CuONPs+α-KG groups. (L) Heatmap displaying downregulated genes in CuONPs-treated blastocyst (compared to the control) and rescued by α-KG supplementation. Data in (C), (D), (G) and (H) are means ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001.

Article Snippet: Afterward, the embryos were permeabilized in a 0.5% Triton X-100 solution for 15 min. Next, the embryos were blocked with 1% BSA (A1933, Sigma), and then were incubated with primary antibodies targeting LC3B (2775s, Cell Signaling Technology), p62 (D5L7G, Cell Signaling Technology), ACO2 (ab110321, Abcam), IDH1 (sc-515396, Santa Cruz), or 5mc (ab214727, Abcam) in 1% BSA.

Techniques: Expressing, Control, RNA Sequencing, Immunofluorescence, Western Blot, Concentration Assay, Transplantation Assay, Staining