Journal: PLoS ONE

Article Title: BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

doi: 10.1371/journal.pone.0084388

Figure Lengend Snippet: ( A-D ) Wild-type, Bim −/− , and Bid −/− MEFs ( panel B ) were exposed to heat shock (44°C for 1–1.5 h) in a humidified incubator (5% CO 2 –95% air). The cells were then transferred to a 37°C incubator and later collected for MOMP (16 h), caspase-3 activation, PARP cleavage (24 h, panel D ), Δψm (24 h, panel C ) and cell death measurements (24 h, panel A ). *p<0.05, significantly different from wild-type cells. ( E-H ) Human Jurkat T cells, stably depleted of BIM or BID by RNAi ( panel F; note: black triangles denote crop marks ), were exposed to heat shock (44°C for 1 h) in a humidified incubator (5% CO 2 –95% air). As before, the cells were then transferred to a 37°C incubator and later collected for MOMP, caspase-3 activation, PARP cleavage (24 h, panel H ), Δψm (24 h, panel G) and cell death measurements (24 h, panel E ). ( I ) Wild-type, Bim −/− , and Bid −/− MEFs were exposed to heat shock as described above, but were left in culture afterwards for 72 h, after which the plates were stained with crystal violet. cPARP, cleaved PARP; cCasp3, cleaved/active caspase-3; S/N, supernatant; Pel, mitochondrial pellet; *p<0.05, significantly different from wild-type cells.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): BAX (CS 2772); BAK (CS 3814); BID (CS 2002), cleaved caspase-3 (CS 9662); total caspase-3 (CS 8G10); hCaspase-9 (CS 9502); β-actin (CS 4970); cytochrome c (CS 11940); PARP (CS 9542); and cleaved PARP (CS 9544).

Techniques: Activation Assay, Stable Transfection, Staining

Journal: PLoS ONE

Article Title: BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

doi: 10.1371/journal.pone.0084388

Figure Lengend Snippet: Wild-type and Bax −/− Bak −/− DKO cells were exposed to ( A-C ) heat shock (44°C for 1–1.5 h) in a humidified incubator (5% CO 2 –95% air), or ( D and E ) UV irradiation (4 min) on a transilluminator. The cells were then transferred to a 37°C incubator and later collected for MOMP (16 h), caspase-3 activation, PARP cleavage (24 h, panels C and E ), Δψm (24 h, panel B ) and cell death measurements (24 h, panels A and D ). cPARP, cleaved PARP; cCasp3, cleaved/active caspase-3; S/N, supernatant; Pel, mitochondrial pellet; *p<0.05, significantly different from wild-type cells.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): BAX (CS 2772); BAK (CS 3814); BID (CS 2002), cleaved caspase-3 (CS 9662); total caspase-3 (CS 8G10); hCaspase-9 (CS 9502); β-actin (CS 4970); cytochrome c (CS 11940); PARP (CS 9542); and cleaved PARP (CS 9544).

Techniques: Irradiation, Activation Assay

Journal: PLoS ONE

Article Title: BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

doi: 10.1371/journal.pone.0084388

Figure Lengend Snippet: ( A-C ) Vector control MEFs and those expressing a DN-caspase-9 (C287A) were exposed to heat shock (44°C for 1–1.5 h) in a humidified incubator (5% CO 2 –95% air). The cells were then transferred to a 37°C incubator and later collected for MOMP (16 h), caspase-3 activation, PARP cleavage (24 h, panel C ), Δψm (24 h, panel B ) and cell death measurements (24 h, panel A ). ( D ) Wild-type, Bim −/− , and DN-caspase-9 MEFs were exposed to heat shock as described above, but were left in culture afterwards for 72 h, after which the plates were stained with crystal violet. cPARP, cleaved PARP; cCasp3, cleaved/active caspase-3; S/N, supernatant; Pel, mitochondrial pellet; *p<0.05, significantly different from vector control cells.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): BAX (CS 2772); BAK (CS 3814); BID (CS 2002), cleaved caspase-3 (CS 9662); total caspase-3 (CS 8G10); hCaspase-9 (CS 9502); β-actin (CS 4970); cytochrome c (CS 11940); PARP (CS 9542); and cleaved PARP (CS 9544).

Techniques: Plasmid Preparation, Expressing, Activation Assay, Staining

Journal: PLoS ONE

Article Title: BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

doi: 10.1371/journal.pone.0084388

Figure Lengend Snippet: ( A ) Cartoon illustrating the interactions of BH3-only proteins with multidomain proapoptotic (BAX and BAK) and antiapoptotic (MCL-1, BCL-2, and BCL-X L ) BCL-2 family members. BH3-only proteins can function as BAX/BAK activators (BIM, tBID, PUMA), or as sensitizers (NOXA, BAD, BMF, BIK, and HRK) that displace activators from antiapoptotic family members. ( B-D ) Wild-type and Mcl-1 −/− MEFs ( panel B, inset ) were exposed to heat shock (44°C for 1–1.5 h) in a humidified incubator (5% CO 2 –95% air). The cells were then transferred to a 37°C incubator and later collected for MOMP (16 h), caspase-3 activation, PARP cleavage (24 h, panel D ), Δψm (24 h, panel C ) and cell death measurements (24 h, panel B ). *p<0.05, significantly different from wild-type cells. ( E and F ) Wild-type MEFs were preincubated with DMSO (control) or ABT-737 (250 nM) for 1 h and subsequently exposed to heat shock (44°C for 1.5 h) in a humidified incubator (5% CO 2 –95% air). The cells were then transferred to a 37°C incubator and later collected for MOMP (16 h), caspase-3 activation, PARP cleavage (24 h, panel F ) and cell death measurements (24 h, panel E ). *p<0.05, cells treated with ABT-737 were significantly different from those treated with DMSO. cPARP, cleaved PARP; cCasp3, cleaved/active caspase-3; S/N, supernatant; Pel, mitochondrial pellet.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): BAX (CS 2772); BAK (CS 3814); BID (CS 2002), cleaved caspase-3 (CS 9662); total caspase-3 (CS 8G10); hCaspase-9 (CS 9502); β-actin (CS 4970); cytochrome c (CS 11940); PARP (CS 9542); and cleaved PARP (CS 9544).

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

doi: 10.1371/journal.pone.0084388

Figure Lengend Snippet: According to the canonical pathway, heat shock induces formation of a RAIDD-caspase-2 complex that activates caspase-2. Following cleavage by caspase-2, truncated Bid (tBID) then stimulates BAX/BAK-dependent MOMP, cytochrome c release, and formation of the Apaf-1/caspase-9 apoptosome complex, which in turn activates the effector caspase-3. In our hands, however, loss of BID or inhibition of the apoptosome provides only modest short-term protection at lower exposures (1 h, 44°C), whereas loss of BIM profoundly inhibits cell death and facilitates long-term protection. In the context of heat shock, BIM induces MOMP and loss of Δψm in a BAX/BAK-dependent manner (and may be responsible for triggering caspase-3 activation in the absence of detectable mitochondrial injury). Thus, BIM appears to mediate an alternative (and perhaps dominant) pathway to heat shock-induced apoptosis.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): BAX (CS 2772); BAK (CS 3814); BID (CS 2002), cleaved caspase-3 (CS 9662); total caspase-3 (CS 8G10); hCaspase-9 (CS 9502); β-actin (CS 4970); cytochrome c (CS 11940); PARP (CS 9542); and cleaved PARP (CS 9544).

Techniques: Inhibition, Activation Assay

Journal: Journal of Virology

Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

doi: 10.1128/JVI.00537-17

Figure Lengend Snippet: L1 protein associated with condensed chromosomes retains the reactivity of the H16.V5 epitope. (A and B) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized, and treated with or without Click-iT reaction buffer without dye. Next, the cells were incubated with L1 conformationally dependent mouse MAb H16.V5 antibody (green) and AF488-conjugated anti-α-tubulin (white). Lastly, the cells were treated with Click-iT reaction buffer with AF555 dye (red) to stain the EdU-labeled pseudogenome and mounted in DAPI (blue). Note that colocalization of the EdU puncta and L1 protein is denoted as a yellow color.

Article Snippet: The microtubule network was detected using mouse MAb anti-α-tubulin conjugated to Alexa Fluor 488 (AF488; catalog no. 8058S; Cell Signaling).

Techniques: Infection, Labeling, Incubation, Staining

Journal: Journal of Virology

Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

doi: 10.1128/JVI.00537-17

Figure Lengend Snippet: L1 protein associated with condensed chromosomes retains the reactivity of the H16.56E epitope. (A and B) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized, and treated with or without Click-iT reaction buffer without dye. Next, the cells were incubated with L1 conformationally dependent mouse MAb H16.56E antibody (green) and AF488-conjugated anti-α-tubulin (white). Lastly, the cells were treated with Click-iT reaction buffer with AF555 dye (red) to stain the EdU-labeled pseudogenome and mounted in DAPI (blue). Note that colocalization of the EdU puncta and L1 protein is denoted as a yellow color.

Article Snippet: The microtubule network was detected using mouse MAb anti-α-tubulin conjugated to Alexa Fluor 488 (AF488; catalog no. 8058S; Cell Signaling).

Techniques: Infection, Labeling, Incubation, Staining

Journal: Journal of Virology

Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

doi: 10.1128/JVI.00537-17

Figure Lengend Snippet: The linear epitope of the 33L1-7 antibody is hidden in the L1 protein associated with condensed chromosomes. (A and B) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized, and treated with or without with Click-iT reaction buffer without dye. Next, the cells were incubated with mouse MAb 33L1-7 antibody (green) and AF488-conjugated anti-α-tubulin (white). Lastly, the cells were treated with Click-iT reaction buffer with AF555 dye (red) to stain the EdU-labeled pseudogenome and mounted in DAPI (blue). Note that colocalization of the EdU puncta and L1 protein is denoted as a yellow color.

Article Snippet: The microtubule network was detected using mouse MAb anti-α-tubulin conjugated to Alexa Fluor 488 (AF488; catalog no. 8058S; Cell Signaling).

Techniques: Infection, Labeling, Incubation, Staining

Journal: Journal of Virology

Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

doi: 10.1128/JVI.00537-17

Figure Lengend Snippet: Some full-length L1 protein accompanies the viral genome to the nucleus. (A) HeLa cells were infected with HPV16 pseudovirus with or without 1.5 μM Eg5 inhibitor III (Eg5i) and tracked via live-cell imaging using the IncuCyte Zoom for 48 h. Note that the images are depicted at 18 hpi. (B) HaCaT cells were infected with EdU-labeled HPV16 pseudovirus in the presence of 1.5 μM Eg5i. The cells were fixed at 24 hpi and permeabilized with either digitonin at 0.625 μg/ml or 0.5% TX-100 and then treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were mounted in DAPI (blue). (C) HaCaT cells were infected with EdU-labeled pseudovirus and treated with 1.5 μM Eg5i. At 24 hpi, the cells were fixed and permeabilized in 0.5% TX-100. Next, the cells were treated with AF555 (red) in Click-iT reaction buffer, followed by incubation with AF488-conjugated anti-α-tubulin (white) and MAb 33L1-7 (green). Lastly, the cells were mounted in DAPI (blue). Note the colocalization between EdU and L1 signal denoted by white arrows. (D) HeLa cells were infected with HPV16 pseudovirus in the presence of 1.5 μM Eg5i. Cells were trypsinized for 2 min, and monoastral cells were collected. Next, the cells were treated with 15 μl of 0.25% trypsin for 1 h at 37°C. The cells were lysed by passage through a 1-ml syringe with a 25-gauge needle 40 times. Cell lysates were incubated for 1 h at 37°C once more, the trypsin was inactivated, and the samples were analyzed by Western blot analysis with a cocktail of HPV16 L1-specific mouse MAbs (IID5, 33L1-7, and 312F).

Article Snippet: The microtubule network was detected using mouse MAb anti-α-tubulin conjugated to Alexa Fluor 488 (AF488; catalog no. 8058S; Cell Signaling).

Techniques: Infection, Live Cell Imaging, Labeling, Incubation, Western Blot

Journal: Nature Communications

Article Title: AIMP2-DX2 provides therapeutic interface to control KRAS-driven tumorigenesis

doi: 10.1038/s41467-022-30149-2

Figure Lengend Snippet: a DX2 levels in H460 lung cancer cells were controlled by the introduction of Strep-DX2 and si-DX2. Protein and mRNA levels were determined by Western blotting (WB) and RT-PCR (RT), respectively. Phosphorylation of ERK and Akt was monitored for KRAS signaling. Actin was used as a loading control. Results are representative of at least three independent experiments. b KRAS level in doxycycline (Dox)-inducible DX2 transgenic mice (Ind-DX2). Levels of KRAS and DX2 in colorectal tissues from two controls (#1, 2) and two Dox-fed mice (#3, 4) were analyzed. c MEF isolated from Ind-DX2 were treated with Dox in a time-dependent manner and protein levels were determined. Results are representative of at least three independent experiments. d Heat map from the results showing DX2-dependent regulation of RAS isoforms. DX2-mediated changes in the levels, stability, and ubiquitination of isoforms were quantified from Supplementary Fig. . Cell viability and anchorage-independent colony formation assay were quantified from Supplementary Fig. . Maximum (max) and minimum (min) values from the indicated experiments were designated as different colors with the highest and lowest intensity, respectively, and the rests were graded according to their relative values. e Result of xenograft ( n = 4) using H460 cells stably expressing the indicated combination of KRAS4B and sh-DX2. Representative images of mice and tumors for each group (left) and tumor sizes (right) were shown. Sh means short hairpin RNA. All error bars represent the standard deviation (S.D.). P value is from the two-sided t test. f Heat map representing the cellular levels of DX2 and KRAS in 18 lung, 12 colorectal and 8 pancreatic cell lines. The cellular levels of DX2 and KRAS in the tested cell lines were represented as the heat map of orange and blue colors, respectively. The maximum and minimum values quantitated as described in Methods were shown with the highest and lowest color intensity, respectively, and the rests were graded according to their relative values. g Analysis of the levels of DX2 and KRAS in lung and colorectal tumor tissues. Staining intensities of the two proteins were classified as low (L) and high (H) (Supplementary Fig. ). Number of the analyzed tissue samples is shown in brackets. h DX2 and KRAS levels in the tumor and matched normal (MN) tissues from 99 patients with colorectal cancer. Depending on the levels in tumor compared to MN, samples were classified as H and L as compared to MN levels (Supplementary Fig. ). Source data are provided as a .

Article Snippet: Primary antibodies against KRAS and DX2 were purchased from Novus Biologicals and CureBio, respectively.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Phospho-proteomics, Control, Transgenic Assay, Isolation, Ubiquitin Proteomics, Colony Assay, Stable Transfection, Expressing, shRNA, Standard Deviation, Staining

Journal: Nature Communications

Article Title: AIMP2-DX2 provides therapeutic interface to control KRAS-driven tumorigenesis

doi: 10.1038/s41467-022-30149-2

Figure Lengend Snippet: a Top 200 proteins identified as the potential binding proteins of DX2 by LC-MS analysis. The relative fold change values of 12 proteins in proliferative ontology were represented as the bar graph (left upper box). Among 12 identified proteins, PRDX1 was detected at the highest fold change and the fold changes of other proteins were divided by that of PRDX1. The relative values of the other eleven proteins were graded, taking the value of PRDX1 as 1. Diameter of circles denotes the relative fold changes of a total of 200 identified proteins. b , c Determination of specific binding of DX2 to KRAS. DX2 was mixed with different nanoluciferase-RAS isoforms. Amounts of the indicated RAS isoforms co-precipitated with DX2 were determined by luciferase activity and are shown as a bar graph ( b ). CCD18CO cells expressing Strep-tagged DX2 and the indicated nanoluciferase-RAS isoforms were precipitated using a strep-tactin column. Amounts of the indicated nanoluciferase-RAS isoforms co-precipitated with DX2 were measured and shown as above ( c ). All the experiments were independently repeated thrice and error bars denote S.D. Data are presented as mean values ± S.D. P value is from the two-sided t test. d In vitro pull-down assay showing direct binding between DX2 and KRAS4B. Co-precipitated KRAS4B or HRAS with GST-DX2 was detected by SDS-PAGE and immunoblotting using an anti-pan RAS antibody. GST proteins were detected by Coomassie staining. Results are representative of at least three independent experiments. e Endogenous KRAS in DLD-1 cells treated with EGF for 30 min was precipitated with the anti-KRAS antibody. p-EGFR was used as a positive control for EGF signaling. Results are representative of at least three independent experiments. f Schematic diagram presenting the binding region between DX2 and KRAS4B. Binding region is depicted as a dashed line (Supplementary Fig. ). g In vitro pull-down assay showing direct interaction between DX2 protein and KRAS4B, but not with HRAS, HVR peptide. Biotin-conjugated and native peptide were serially mixed with DX2 proteins for checking the binding specificity. Results are representative of at least three independent experiments. EV, PD, IP, and WCL represent empty vector, pull-down, immunoprecipitation, and whole cell lysate, respectively. Source data are provided as a .

Article Snippet: Primary antibodies against KRAS and DX2 were purchased from Novus Biologicals and CureBio, respectively.

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Luciferase, Activity Assay, Expressing, In Vitro, Pull Down Assay, SDS Page, Western Blot, Staining, Positive Control, Plasmid Preparation, Immunoprecipitation

Journal: Nature Communications

Article Title: AIMP2-DX2 provides therapeutic interface to control KRAS-driven tumorigenesis

doi: 10.1038/s41467-022-30149-2

Figure Lengend Snippet: a , b Chemical shift perturbation of 15 N-labeled DX2 51-251 (C136S, C222S) by the binding to KRAS4B HVR peptide. Superposition of the 2-dimensional (2D) 1 H- 15 N TROSY spectra of free DX2 in the presence (red) and absence (black) of HVR peptide. Strongly affected residues are depicted ( a , left) and the indicated signals are enlarged and presented ( a , right). The residues showing strong (Δδ > 0.02 ppm) and medium (0.01 < CSP ≤ 0.02 ppm) perturbation are shown in red and orange, respectively, on the surface of the DX2 GST domain ( b ). c Result of 600 ns molecular dynamics (MD) simulation to determine the binding surface of DX2 and KRAS4B. The two significant interfaces are highlighted by the dashed box. d Significant residues for binding between DX2 and KRAS4B were analyzed by immunoprecipitation using alanine mutants (Supplementary Fig. ) and presented in the enlarged image of each interface. e , f Validation of the effect of KRAS4B farnesylation on its binding with DX2. H460 cells expressing Strep-DX2 were treated FTase inhibitor I and separated into cytosol and membrane fraction. HSP90 and cadherin (Cad) were used as loading markers for cytosol (Cyto) and membrane (Memb), respectively ( e ). In vitro pull-down assay showing the binding of DX2 to farnesylated KRAS4B. In vitro farnestlyated KRAS4B proteins were mixed with the purified GST-DX2. Binding of the two proteins and farnesylation were determined by immunoblotting and fluorescent scans, respectively. N and F indicate native and farnesylation, respectively ( f ). Results are representative of at least three independent experiments. g H460 cells introducing Strep-DX2 were treated with EGF and separated into Cyto and Memb fractions. Relative levels of KRAS were presented below the KRAS blots. Results are representative of at least three independent experiments. h Schematic presentation for binding between DX2 and KRAS. un KRAS and ST KRAS mean unstable and stable KRAS, respectively. Source data are provided as a .

Article Snippet: Primary antibodies against KRAS and DX2 were purchased from Novus Biologicals and CureBio, respectively.

Techniques: Labeling, Binding Assay, Immunoprecipitation, Biomarker Discovery, Expressing, Membrane, In Vitro, Pull Down Assay, Purification, Western Blot

Journal: Nature Communications

Article Title: AIMP2-DX2 provides therapeutic interface to control KRAS-driven tumorigenesis

doi: 10.1038/s41467-022-30149-2

Figure Lengend Snippet: a Smurf2-mediated levels of KRAS. Endogenous levels of KRAS from H460 cells expressing Smurf2 wild type (WT), catalytic-inactive mutant (CA), and si-Smurf2 were determined by immunoblotting. si means siRNA. b Smurf2-mediated ubiquitination of KRAS. The cells described in ( a ) were treated with MG-132 and the ubiquitinated amounts of KRAS were analyzed by ubiquitination assay using the anti-ubiquitin (Ub) antibody. c In vitro ubiquitination assay showing the effect of DX2 on Smurf2-mediated ubiquitination of KRAS4B. UBE1, UbcH5c/UBE2D3, Smurf2, and KRAS4B were used as E1, E2, E3, and substrate for the reaction, respectively. Proteins were confirmed by Coomassie staining in the input panel (bottom). Ubiquitination of KRAS4B was detected by immunoblotting as above (upper). d H460 cells expressing si-Smurf2 and Strep-DX2 were subjected to immunoblotting. Quantitative levels of KRAS were depicted below its blot. e In vitro pull-down assay showing the effect of DX2 on the binding of Smurf2 to KRAS4B. Smurf2, GST-KRAS4B, and DX2 proteins were mixed, and co-precipitation of DX2 and Smurf2 with KRAS4B was monitored. f Inhibitory effect of DX2 on Smurf2-mediated ubiquitination of KRAS4B. 293T cells expressing GFP-KRAS4B, FLAG-Smurf2, and Strep-DX2 were treated with MG-132 and separated into cytosol and membrane fractions. g Analysis of binding kinetics for DX2 or Smurf2 to KRAS (left), and KRAS, HSP70 or p14ARF to DX2 (right) upon EGF signal. All the interactions analyzed by immunoprecipitation (Supplementary Fig. ) were quantified and presented as graphs. C and N indicate cytosol and nucleus, respectively. The maximum blot intensities obtained from immunoprecipitation of each protein pair was taken as 1, and the other blot intensities were divided by the maximum intensity values and presented as the relative values. h Ubiquitination assay validating the Smurf2-dependent ubiquitination sites of KRAS4B. H460 cells expressing GFP-KRAS4B mutants and FLAG-Smurf2 were treated with MG-132 and subjected to the ubiquitination assay. Source data are provided as a . a – f , h , Results are representative of at least three independent experiments.

Article Snippet: Primary antibodies against KRAS and DX2 were purchased from Novus Biologicals and CureBio, respectively.

Techniques: Expressing, Mutagenesis, Western Blot, Ubiquitin Proteomics, In Vitro, Staining, Pull Down Assay, Binding Assay, Membrane, Immunoprecipitation

Journal: Nature Communications

Article Title: AIMP2-DX2 provides therapeutic interface to control KRAS-driven tumorigenesis

doi: 10.1038/s41467-022-30149-2

Figure Lengend Snippet: a Structure of BC-DXI-32982 (DXI hereafter). b Inhibitory effect of DXI on the binding of DX2 and KRAS4B. Binding pair of PRKACA and PRKAR2A was used for checking the binding specificity. N.D. denotes “not determined” in the tested range of dose. The experiment was independently repeated thrice and error bars denote S.D. Data are presented as mean values ± S.D. P value is from the two-sided t test. c Endogenous immunoprecipitation showing the interaction between DX2 and KRAS in H460 cells treated with DXI. Results are representative of at least three independent experiments. d In vitro pull-down assay showing direct inhibition of DXI on the binding of DX2 and KRAS4B. DXI was incubated with the mixture of cellular extracts expressing GFP-KRAS4B and the purified GST-DX2 proteins. Results are representative of at least three independent experiments. e Suppression of KRAS level via DXI. H460 cells treated with various concentration of DXI for 18 h were subjected to immunoblotting and RT-PCR. Results are representative of at least three independent experiments. f Ubiquitination assay analyzing DXI-mediated ubiquitination of KRAS. Results are representative of at least three independent experiments. g Heat map showing EC 50 of DXI on lung, colorectal, and pancreatic cancer cell lines expressing different levels of DX2 (Supplementary Fig. ). In each type of cancers, the maximum and minimum EC 50 values were represented as blue color with the highest and lowest intensity, respectively, and the rests were graded according to their relative values. h , i In vivo efficacy of DXI on tumor growth. Mice ( n = 3) xenografted with H460 cells were intraperitoneally injected with DXI (1 or 5 mg/kg) five times in 12 d ( h ). Mice ( n = 6) xenografted with HCC1588 or A549 cells were intravenously injected with DXI (5 mg/kg) five times during the experiment ( i ). All error bars represent the standard deviation (S.D.). P value is from the two-sided t test. Source data are provided as a .

Article Snippet: Primary antibodies against KRAS and DX2 were purchased from Novus Biologicals and CureBio, respectively.

Techniques: Binding Assay, Immunoprecipitation, In Vitro, Pull Down Assay, Inhibition, Incubation, Expressing, Purification, Concentration Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Ubiquitin Proteomics, In Vivo, Injection, Standard Deviation

Journal: Nature Communications

Article Title: AIMP2-DX2 provides therapeutic interface to control KRAS-driven tumorigenesis

doi: 10.1038/s41467-022-30149-2

Figure Lengend Snippet: a In vitro pull-down assay showing the direct binding of DXI with DX2, but not with KRAS4B. Proteins co-precipitated with Biotin-DXI #2 (Bio-DXI) were determined by SDS-PAGE and Coomassie staining. Biotin was used as a negative control. Results are representative of at least three independent experiments. b In vitro pull-down assay showing the specific interaction of compound with DX2 using Bio-DXI and DXI. Results are representative of at least three independent experiments. c Binding mode of DXI to DX2 as obtained from the MD simulation. The representative structure at 371.2 ns is shown as an electrostatic surface model (first from above). Zoomed-in view of the detailed interactions is presented and binding residues of the compound (green) were depicted as a stick model (second from above). MD snapshots of DX2 and KRAS4B complex at 533.7 ns (third from above). Overlay of above two MD snapshots by superimposing over DX2. The dashed circle denotes the contact region proposed to be responsible for the DXI-mediated inhibition of the interaction of DX2 with KRAS4B (bottom). d Graphs showing the critical residues of DX2 for binding with DXI. Results from the in vitro binding of DX2 mutants with DXI; compound-mediated change in KRAS levels and inhibition of DX2 mutants-KRAS4B were quantified (Supplementary Fig. ). e Anchorage-independent colony formation assay validating the significance of the binding between DXI and DX2. Representative images are shown (bottom). The experiment was independently repeated thrice and error bars denote S.D. Data are presented as mean values ± S.D. P value is from the two-sided t test. Source data are provided as a .

Article Snippet: Primary antibodies against KRAS and DX2 were purchased from Novus Biologicals and CureBio, respectively.

Techniques: In Vitro, Pull Down Assay, Binding Assay, SDS Page, Staining, Negative Control, Inhibition, Colony Assay

Journal: Cell reports

Article Title: iRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation

doi: 10.1016/j.celrep.2021.109642

Figure Lengend Snippet: RESOURCE TABLE

Article Snippet: Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal anti-RPS2 (uS5) Bethyl Laboratories Cat# A303-794A; RRID:AB_11218192 Rabbit monoclonal anti-RPS3 (uS3) Bethyl Laboratories Cat# A303-840A; RRID:AB_2615588 Rabbit polyclonal anti-RPS10 (eS10) ABclonal Cat# A6056; RRID:AB_2766730 Rabbit monoclonal anti-RPS20 (uS10_ Abcam Cat# ab133776; RRID:AB_2714148 Rabbit monoclonal anti-p-eIF2 α Cell Signaling Technology Cat# 3398S; RRID:AB_2096481 Rabbit polyclonal anti-RPL7 (uL30) Bethyl Laboratories Cat# A300-741A; RRID:AB_2301241 Rabbit polyclonal anti-USP10 Abcam Cat# ab72486; RRID:AB_1271412 Mouse monoclonal α-Tubulin Cell Signaling Technology Cat# 3873; RRID:AB_1904178 Mouse monoclonal c-Myc Santa Cruz Biotechnology Cat# sc-40 AC; RRID:AB_2857941 Mouse monoclonal anti-tubulin Cell Signaling Technology Cat# 3873S; RRID:AB_1904178 Anti-Rabbit IgG (H+L), HRP Conjugate antibody Promega Cat# W4011; RRID:AB_430833 Anti-Mouse IgG (H+L), HRP Conjugate antibody Promega Cat# W4021; RRID:AB_430834 Lipofectamine RNAiMax Thermo-Fisher Cat# 13778030 Lipofectamine 2000 THermo-Fisher Cat# 11668019 Mirus TransIT 293 Mirus Bio llc Cat# MIR 2700 Protease inhibitor cocktail tablet Roche Cat# 11836170001 Immun-Blot® PVDF Membrane BioRad Cat# 1620177 Albumin Standard Thermo Scientific Cat# 23209 Clarity™ Western ECL Substrate BioRad Cat# 170-5061 Chemicals DL-Dithiothreitol ACROS organics Cat# 165680050 Anisomycin Fisher Scientific Cat# 50995788 Harringtonine LKT labs Cat# H0169 Thapsigargin Tocris Biosciences Cat# 1138 Cycloheximide MP Biomedicals Cat# 100183 Sodium arsenite solution Fluka Analytical Cat# 35000-1L-R Torin1 Tocris Biosciences Cat# 4247 PatamineA Gift from Jeremy Pelletier’s lab MG-132 Enzo Life Sciences Cat# BML-PI102 Rocaglamide MedChemExpress Cat# HY19356 Lactimidomycin Millipore Sigma Cat# 5062910001 Bafilomycin A Sigma Cat# B1793 SAR405 APExBio Cat# A8883 Doxycycline hydrochloride Fisher Scientific Cat# BP2653-5 Ammonium persulfate (≥ 98%) Sigma Cat# A3678 Fetal Bovine Serum VWR Cat# 97068-085 Trypsin Sigma-Aldrich Cat# T1426 N-Ethylmaleimide Sigma-Aldrich Cat# E3876 Puromycin Mediatech (Corning) Cat# 61-385-RA RNaseA Thermo Scientific Cat# EN0531 SUPERase-In™ Invitrogen Cat# AM2696 Turbo™ DNase Invitrogen Cat# AM2238 Trizol Life Technologies Cat# 15596026 Dimethyl sulfoxide Corning Cat# 25-950-CQC β-mercaptoethanol J.T.

Techniques: Protease Inhibitor, Western Blot, Bicinchoninic Acid Protein Assay, Bradford Assay, Recombinant, Sequencing, Software

Journal: Cell reports

Article Title: iRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation

doi: 10.1016/j.celrep.2021.109642

Figure Lengend Snippet: RESOURCE TABLE

Article Snippet: Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal anti-RPS2 (uS5) Bethyl Laboratories Cat# A303-794A; RRID:AB_11218192 Rabbit monoclonal anti-RPS3 (uS3) Bethyl Laboratories Cat# A303-840A; RRID:AB_2615588 Rabbit polyclonal anti-RPS10 (eS10) ABclonal Cat# A6056; RRID:AB_2766730 Rabbit monoclonal anti-RPS20 (uS10_ Abcam Cat# ab133776; RRID:AB_2714148 Rabbit monoclonal anti-p-eIF2 α Cell Signaling Technology Cat# 3398S; RRID:AB_2096481 Rabbit polyclonal anti-RPL7 (uL30) Bethyl Laboratories Cat# A300-741A; RRID:AB_2301241 Rabbit polyclonal anti-USP10 Abcam Cat# ab72486; RRID:AB_1271412 Mouse monoclonal α-Tubulin Cell Signaling Technology Cat# 3873; RRID:AB_1904178 Mouse monoclonal c-Myc Santa Cruz Biotechnology Cat# sc-40 AC; RRID:AB_2857941 Mouse monoclonal anti-tubulin Cell Signaling Technology Cat# 3873S; RRID:AB_1904178 Anti-Rabbit IgG (H+L), HRP Conjugate antibody Promega Cat# W4011; RRID:AB_430833 Anti-Mouse IgG (H+L), HRP Conjugate antibody Promega Cat# W4021; RRID:AB_430834 Lipofectamine RNAiMax Thermo-Fisher Cat# 13778030 Lipofectamine 2000 THermo-Fisher Cat# 11668019 Mirus TransIT 293 Mirus Bio llc Cat# MIR 2700 Protease inhibitor cocktail tablet Roche Cat# 11836170001 Immun-Blot® PVDF Membrane BioRad Cat# 1620177 Albumin Standard Thermo Scientific Cat# 23209 Clarity™ Western ECL Substrate BioRad Cat# 170-5061 Chemicals DL-Dithiothreitol ACROS organics Cat# 165680050 Anisomycin Fisher Scientific Cat# 50995788 Harringtonine LKT labs Cat# H0169 Thapsigargin Tocris Biosciences Cat# 1138 Cycloheximide MP Biomedicals Cat# 100183 Sodium arsenite solution Fluka Analytical Cat# 35000-1L-R Torin1 Tocris Biosciences Cat# 4247 PatamineA Gift from Jeremy Pelletier’s lab MG-132 Enzo Life Sciences Cat# BML-PI102 Rocaglamide MedChemExpress Cat# HY19356 Lactimidomycin Millipore Sigma Cat# 5062910001 Bafilomycin A Sigma Cat# B1793 SAR405 APExBio Cat# A8883 Doxycycline hydrochloride Fisher Scientific Cat# BP2653-5 Ammonium persulfate (≥ 98%) Sigma Cat# A3678 Fetal Bovine Serum VWR Cat# 97068-085 Trypsin Sigma-Aldrich Cat# T1426 N-Ethylmaleimide Sigma-Aldrich Cat# E3876 Puromycin Mediatech (Corning) Cat# 61-385-RA RNaseA Thermo Scientific Cat# EN0531 SUPERase-In™ Invitrogen Cat# AM2696 Turbo™ DNase Invitrogen Cat# AM2238 Trizol Life Technologies Cat# 15596026 Dimethyl sulfoxide Corning Cat# 25-950-CQC β-mercaptoethanol J.T.

Techniques: Protease Inhibitor, Western Blot, Bicinchoninic Acid Protein Assay, Bradford Assay, Recombinant, Sequencing, Software

Journal: bioRxiv

Article Title: Stem cell-derived cranial and spinal motor neurons reveal proteostatic differences between ALS resistant and sensitive motor neurons

doi: 10.1101/629295

Figure Lengend Snippet: (A) Experimental outline: motor neurons were differentiated and treated with DMSO or 100 nM MG-132 for 18 h on Day 4 followed by western blot analysis on Day 5. (B) Western blot analysis of Day 5 DMSO or MG-132 treated iCrMNs and iSpMNs using ubiquitin antibody revealed a smear of ubiquitinated proteins in the soluble (RIPA) and insoluble (urea) fractions. iCr, iCrMN; iSp, iSpMN. (C) Quantification of accumulation of ubiquitinated proteins between MG-132 and DMSO treated neurons (n=3, mean ± SD). (D) Western blot analysis of Day 5 DMSO or MG-132 treated iCrMNs and iSpMNs using p62 antibody. Accumulation of p62 proteins is shown in soluble (RIPA) and insoluble (urea) fractions. iCr, iCrMN; iSp, iSpMN. (E) Quantification of p62 accumulation between MG-132 and DMSO treated neurons (n=3, mean ± SD). β-actin in the soluble fraction was used as a loading control for normalization in (C) and (E). The dashed line represents equal accumulation, y=1. n = biological replicates; statistical analysis was performed by Two-way ANOVA and student’s t-test, * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Primary antibodies used for Western Blot: rabbit polyclonal antibody to HA tag (1:5000; Abcam, ab9110); mouse monoclonal antibody to SQSTM1/p62 (1:5000; Abcam, ab56416); rabbit polyclonal antibody to Superoxide Dismutase 1 (SOD1) (1:1000; Abcam, ab16831); mouse monoclonal antibody to ubiquitin (1:5000; CST, #3936); mouse monoclonal antibody to beta-actin (1:5000; Santa Cruz, sc-47778).

Techniques: Western Blot