Journal: Molecular Biology of the Cell

Article Title: Host cell perforation by listeriolysin O (LLO) activates a Ca 2+ -dependent cPKC/Rac1/Arp2/3 signaling pathway that promotes Listeria monocytogenes internalization independently of membrane resealing

doi: 10.1091/mbc.E17-09-0561

Figure Lengend Snippet: Rac1, RhoA, and Cdc42 knockdown efficiencies. (A, B) Western blot analysis of Rac1, RhoA, and Cdc42 in nontreated HepG2 cells and in HepG2 cells treated with a negative control siRNA (NC) or with Rac1-, RhoA-, or Cdc42-specific siRNAs. (A) Western blot analysis of cell lysates (nondiluted, 1:2, 1:4, and 1:8 diluted) using anti-Rac1, -RhoA, -Cdc42, and -actin antibodies. (B) Western blot analysis of nondiluted cell lysates to verify the absence of compensatory expression of Rac1 (top), RhoA (middle), or Cdc42 (bottom) in cells treated with Rac1-, RhoA-, and Cdc42-siRNAs. All Western blots are representative of at least three independent experiments.

Article Snippet: Antibodies include the following: anti– L. monocytogenes rabbit polyclonal antibodies (GeneTex); goat anti-rabbit secondary antibodies conjugated to Alexa488 and Alexa568 (Molecular Probes); anti-BSA rabbit antiserum (B1520; Sigma-Aldrich); mouse monoclonal antibodies directed against Rac1, RhoA, and Cdc42 (clones ARC03, ARH04, and ACD03, respectively; Cytoskeleton); rabbit anti-actin (clone A2103; Sigma-Aldrich); and secondary goat anti-mouse immunoglobulin G (IgG) and goat anti-rabbit IgG antibodies conjugated to horseradish peroxidase (Cell Signaling).

Techniques: Western Blot, Negative Control, Expressing

Journal: Molecular Biology of the Cell

Article Title: Host cell perforation by listeriolysin O (LLO) activates a Ca 2+ -dependent cPKC/Rac1/Arp2/3 signaling pathway that promotes Listeria monocytogenes internalization independently of membrane resealing

doi: 10.1091/mbc.E17-09-0561

Figure Lengend Snippet: Role of Rac1 in LLO-dependent entry of L. monocytogenes . (A, B) Nontreated HepG2 cells and HepG2 cells treated with negative control (NC), Rac1-, RhoA-, and Cdc42-siRNAs were incubated with WT (10403s) or the LLO deficient ( Δhly ) L. monocytogenes (A), or with BSA or BSA/LLO-coated beads (B), at a multiplicity of infection 20 (MOI 20) for 30 min at 37°C. (C) HepG2 cells expressing mCit-Rac1 or dominant negative mCit-Rac1N17 were incubated with BSA/LLO-coated beads at MOI 5 for 30 min at 37°C. Cells were then fixed and bacteria or beads were fluorescently labeled to enumerate the total number of bacteria (Nt) and the number of extracellular bacteria associated with host cells (Ne) (A, B, C). Entry efficiency was measured as 100 × [(Nt)-(Ne)]/(Nt) and results are expressed as the percentage entry ± SEM relative to NC-siRNA-treated cells incubated with WT (A) or BSA/LLO-coated polystyrene beads (B) ( n ≥ 3). In C, results are expressed as the percentage entry ± SEM relative to mCit-Rac1 expressing cells incubated with BSA/LLO-coated beads ( n = 4). Statistics are as follows: * p < 0.05; ** p < 0.01; *** p < 0.005.

Article Snippet: Antibodies include the following: anti– L. monocytogenes rabbit polyclonal antibodies (GeneTex); goat anti-rabbit secondary antibodies conjugated to Alexa488 and Alexa568 (Molecular Probes); anti-BSA rabbit antiserum (B1520; Sigma-Aldrich); mouse monoclonal antibodies directed against Rac1, RhoA, and Cdc42 (clones ARC03, ARH04, and ACD03, respectively; Cytoskeleton); rabbit anti-actin (clone A2103; Sigma-Aldrich); and secondary goat anti-mouse immunoglobulin G (IgG) and goat anti-rabbit IgG antibodies conjugated to horseradish peroxidase (Cell Signaling).

Techniques: Negative Control, Incubation, Infection, Expressing, Dominant Negative Mutation, Labeling

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: CPAP depletion does not affect Rab5 recruitment to the endosomes and ligand-bound EGFR-positive endosomes HeLa cells expressing control or CPAP shRNA were treated with AF555-EGF ligand for the indicated time points, stained, and subjected to 4-color imaging by confocal microscopy. Images representing the detection of Rab5 and EEA1 (A), Rab5 and CD63 (B), and Rab5 (along with AF555-EGF) (C) are shown. Left (A–C): maximum intensity-projection images of confocal Z stacks. Right (A–C): single Z-plane of images. Bottom left graph in (C): co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing Rab5 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Bottom right graph in (C): relative integrated fluorescence intensity values of Rab5 staining quantified in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Scale bars: 10 μm. p values were not statistically significant. Zoomed images correspond to the dashed inset boxes of the indicated images. Note: (B) and (C) present data from the same experiments where 4-color imaging was done and convey information on two different aspects based on three markers at a time. Since the visuals of the same cell can help with more reliable interpretation of the data, images of the same cell were used, where possible, for (B) and (C) with the same or different pseudo-color. Hence, duplication of some sub-images among (B) and (C) is intentional.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Expressing, Control, shRNA, Staining, Imaging, Confocal Microscopy, Fluorescence

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: CPAP depletion disrupts Rab5-to-Rab7 conversion (A) HeLa cells expressing control or CPAP shRNA were treated with untagged EGF for 60 min, stained for Rab5 and Rab7, and imaged by Airyscan super-resolution microscopy. Left: maximum intensity-projection images of Z stacks; middle: single Z-plane of images; right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7 (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. (B) HeLa cells expressing control or CPAP shRNA were transfected with GFP-Rab5 and mCherry-Rab7 constructs, treated with untagged EGF for 60 min, and imaged by Airyscan super-resolution microscopy. Left: maximum intensity-projection images of Z stacks; middle: single Z-plane of images; right: co-localization (yellow) was quantified by determining the percentages of GFP-Rab5-positive (green) puncta containing mCherry-Rab7 (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. The object-based co-localization macro tool FIJI was employed. (C) HeLa cells stably expressing control or CPAP-shRNA were transfected with control vector (GFP), GFP-Rab7 (WT; wild-type) or GFP-Rab7 (DN; dominant negative) vector constructs, treated with AF555-EGF ligand for 60 min, and stained for CD63 to mark MVBs/late endosomes, and imaged by confocal microscopy to determine AF555-EGF and CD63 co-localization. Left top row: GFP expression in control and Rab7 construct-expressing cells; left bottom rows: representative single Z-plane of images showing ligand-bound EGFR-CD63 co-localization; right: co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing CD63 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Expressing, Control, shRNA, Staining, Super-Resolution Microscopy, Transfection, Construct, Stable Transfection, Plasmid Preparation, Dominant Negative Mutation, Confocal Microscopy, MANN-WHITNEY

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: Rab5-to-Rab7 conversion and EGFR trafficking to late endosomes are restored in CPAP-depleted cells upon HRS, but not TSG101, overexpression (A) Schematic of the experimental strategy using control and CPAP-specific siRNA-treated HeLa cells with and without GFP-HRS or GFP-TSG101 expression. (B and C) Control and CPAP-specific siRNA-treated HeLa cells were subjected to mock or GFP-HRS or GFP-TSG101 vector transfection for 24 h, treated with untagged EGF for 60 min, and stained for Rab5 and Rab7 (B) or treated with AF555-EGF for 30 min and stained for Rab7 (C) and imaged by Lightning super resolution microscopy. Left: representative single Z-plane of images showing localization of Rab7 on Rab5-positive puncta (B) and AF555-EGF on Rab7-positive puncta (C) in cells with and without GFP-HRS or GFP-TSG101 expression. Right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7-positive (red) puncta in (B) and EGF-positive (red) puncta containing Rab7 (green, pseudo-color) in (C) in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗<0.05, ∗∗ <0.01, ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Over Expression, Control, Expressing, Plasmid Preparation, Transfection, Staining, Super-Resolution Microscopy, MANN-WHITNEY

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: Rab5-to-Rab7 conversion and EGFR trafficking to MVB are restored in CPAP- and HRS-depleted cells upon exogenous CPAP expression (A) Schematic of the experimental strategy using control and CPAP- and HRS-specific siRNA-treated HeLa cells with and without siRNA-resistant GFP-CPAP expression. (B) Airyscan super-resolution microscopy images showing cells stained for Rab5 and Rab7 at 60 min time point. Left: representative single Z-plane of images showing localization of Rab5- and Rab7-positive puncta in cells with and without GFP expression. Right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7-positive (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. (C) Confocal microscopy images showing cells stained for CD63 and AF555-EGF at 60 min time point. Left: representative single Z-plane of images showing localization of CD63 − and EGF-positive puncta in cells with and without GFP expression. Right: co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing CD63-positive (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. The object-based co-localization macro tool FIJI was employed for (B) and (C). Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗∗∗<0.001, ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Expressing, Control, Super-Resolution Microscopy, Staining, Confocal Microscopy, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: a Polar interactions formed by Arg 249 with neighbouring residues in p53 WT . b Change in polar contacts with neighbouring residues due to mutation of Arg 249 to serine in p53 R249S . b Superimposed view of the mutation site in p53 WT and p53 R249S . Residues of p53 WT are shown in green and for p53 R249S they are shown in pink. Change in the structure at 249th position is highlighted by a circle. Residues that acquired significantly different conformations are shown in stick representation. c Root mean square deviations of residues in p53 WT and p53 R249S . (a) RMSD of Arg 248 during the course of MD simulation in p53 WT (green) and in p53 R249S (red). (b) RMSD of residues from 249 to 271 in p53 WT (green) and p53 R249S (red)

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Mutagenesis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: a Difference in the molecular surface near Y220 position in p53 WT and p53 Y220C mutant. a Size of cavity in p53 WT . b Mutation from Tyr to Cys at 220th position rearranges the conformation of surrounding residues, especially located in the loop region. Widening of the loop near 220th position and removal of the Tyr side chain creates a larger cavity in p53 Y220C protein. c Superimposition of the two molecular surfaces reveals the stringency of the p53 WT (green color) as compared to the p53 Y220C (red color). b Water network near 220th residue in p53 WT and p53 Y220C protein. a Tyr 220 stabilizes the residues of surrounding loops with the help of water molecules. b Cys 220 stabilizes the cavity by solvating it with water molecules. Binding of the withanolides with p53 WT near Tyr 220. Wi-A c and Wi-N d were found to interact with the surface residues near Tyr 220 as no deep cavity was present in p53 WT

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Mutagenesis, Residue, Binding Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: Interactions of Wi-A with p53 Y220C near Cys 220. a Binding pose of Wi-A within the binding site near Cys 220 b 2D representation of the interactions of Wi-A with p53 Y220C . Interactions of Wi-N with p53 Y220C protein structure. c Binding pose of Wi-N within the binding cavity of p53 Y220C near Cys 220 d 2D representation of the interactions of Wi-N with surrounding residues within the cavity of p53 Y220C . Charge complementarity of Wi-A and Wi-N with the binding cavity. Wi-A e and Wi-N f both were fitting inside the cavity according to the charge distribution within it. White region represents the hydrophobic region whereas blue and red represents the hydrophilic region

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Binding Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: Wi-A furnished wild type p53 function in mutant p53 (p53 Y220C ) horboring hepatoma cells. a Viability assay of human hepatocarcinoma with wild type p53 (HuH-6), mutant p53 (HuH-7), and telomerized human cells bearing p53 mutants (p53 V143A , p53 R249S and p53 R273H ). b Comparison of response of HuH-6 and HuH-7 cells to Wi-A. Dose response was observed for both the cell lines. HuH-7 showed stronger cytotoxicity to Wi-A. c Western blot showed reduction in mortalin and increase in p53 in cells treated with 1 μM Wi-A in the p53 mutants, p53 V143A and p53 R273H ; p53 R249S cells possessed low expression that remained undetected on these blots. In contrast to increase in the expression of p53 V143A and p53 R273H , mutant p53 Y220C protein expression was decreased in Wi-A treated cells. d Dose dependent decrease in mutant p53 Y220C protein expression in Wi-A treated cells. e Immunostaining of mortalin and p53 (40 x magnification) in control and Wi-A (0.5 μM) showing increase in nuclear p53 V143A and p53 R273H . HuH-6 (p53 WT ) cells showed increase in nuclear p53 staining. In contrast, HuH-7 (p53 Y220C ) cells exhibited decrease in p53 nuclear staining

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Mutagenesis, Viability Assay, Comparison, Western Blot, Expressing, Immunostaining, Control, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: Wi-A induced restoration of wild type p53 and induction of senescence in HuH-7 cells. a Wild type p53 reporter activity in mock (untransfected), control (transfected and untreated) and Wi-A (transfected and Wi-A treated) cells. Luciferase reporter assays driven either by p53 consensus binding sites (PG13-Luc) or by a p21 WAF-1 promoter (WWP) showed an increase in Wi-A treated cells. b Flow cytometry analysis revealed G 2 cell cycle phase arrest in HuH-7 cells. c Immunostaining of p21 WAF-1 in HuH-6 and HuH-7 control and Wi-A treated cells showing increase in p21 WAF-1 expression in the latter. d Senescence-associated β-galactosidase staining was observed in Wi-A treated HuH-6 and HuH-7 cells (10 x phase magnification). e Wi-A treated HuH-6 and HuH-7 cells showed enhanced staining for nuclear heterochromatin protein HP1γ

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Activity Assay, Control, Transfection, Luciferase, Binding Assay, Flow Cytometry, Immunostaining, Expressing, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: Wi-A induced apoptosis of HuH-7 cells. a Annexin-V staining revealed induction of early apoptosis in Wi-A treated cells. b Apoptosis in Wi-A treated HuH-7 cells was marked by cleavage of caspase3, increase in p21 WAF-1 and phosphorylated p53. c Wi-A treated cells showed Single Strand Breaks as determined by a comet assay, 40 X magnification. d Wi-A rich extract (AL-βCD) treated HuH-6 (p53 WT ) and HuH-7 (p53 Y220C ) cells showed increase and decrease in nuclear p53, respectively. e Increase in the expression of p21 WAF-1 was observed in AL-βCD treated HuH-6 and HuH-7 cells. f HuH-7 cells showed strong cytotoxicity to AL-βCD

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Staining, Single Cell Gel Electrophoresis, Expressing

Journal: Nature Communications

Article Title: Rab27a regulates the transport of influenza virus membrane proteins to the plasma membrane

doi: 10.1038/s41467-025-61587-3

Figure Lengend Snippet: a A549 cells were transfected with siNC or siRNAs targeting Rab3b (siRab3b-1, siRab3b-2), Rab6a (siRab6a-1, siRab6a-2), Rab11a (siRab11a-1, siRab11a-2), Rab17 (siRab17-1, siRab17-2), Rab23a (siRab23a-1, siRab23a-2), Rab25 (siRab25-1, siRab25-2), Rab27a (siRab27a-1, siRab27a-2), Rab37 (siRab37-1, siRab37-2), or Rab38 (siRab38-1, siRab38-2) for 24 h. The cells were then infected with HM virus (MOI, 10), and HA protein levels on the cell surface were quantified by flow cytometry at 4 hpi. b A549 cells transfected with siNC or siRab27a (siRab27a-1, siRab27a-2) for 24 h were infected with HM virus (MOI, 0.1). c–e Rab27a KO and A549-Cas9 cells were infected with HM virus (MOI, 0.1), SH13/H9N2 virus (MOI, 0.01), or PR8/H1N1 virus (MOI, 0.01). f–h A549 cells were transfected with 2 μg/ml exogenous Rab27a or an empty vector as a negative control for 24 h. The cells were then infected with HM virus (MOI, 0.1), SH13 virus (MOI, 0.01), or PR8 virus (MOI, 0.01). i A549-Cas9, Rab27a KO, and Rab27a KO cells stably expressing Flag-Rab27a-WT, Flag-Rab27a-T23N (dominant-negative mutant), or Flag-Rab27a-Q78L (constitutively active mutant) were infected with HM virus (MOI, 0.1). Rab27a and NP protein expression levels were examined by western blot ( b , f , g , h , and i ), with GAPDH or β-actin serving as a loading control. Viral titers in the supernatants were quantified using a TCID 50 assay on MDCK cells ( b–i ). Data represent the means ± SD from three independent experiments. Statistical significance was determined using a two-tailed Student’s t -test.

Article Snippet: The antibodies used in this study and their sources are as follows: Rabbit polyclonal antibodies: Rab37, Rab3b, Rab6a, Rab11a, Rab17, Rab23, Rab25, Rab27a, Rab27b, SYTL4, SQSTM1, ATB4B, TNG46, GM130 (Proteintech; 13051-1-AP, 15774-1-AP, 10187-2-AP, 20229-1-AP, 17501-1-AP, 11101-1-AP, 13189-1-AP, 16868-1-AP, 13412-1-AP, 12128-1-AP, 18420-1-AP, 15131-1-AP, 13573-1-AP, 11308-1-AP), Rab38 (Abways, AY3825), SYTL1 (Bethyl, A305-648A-T), BECN1 (ABclonal Biotechnology, A7353), Phospho-ATG4B (Ser316) (Affinity Biosciences, AF3505), ATG16L1 (HUABIO, ET7106-65), and IAV M2, NP, NS1, and HA (GeneTex; GTX125951, GTX30852, GTX125990, GTX127357).

Techniques: Transfection, Infection, Virus, Flow Cytometry, Plasmid Preparation, Negative Control, Stable Transfection, Expressing, Dominant Negative Mutation, Mutagenesis, Western Blot, Control, Two Tailed Test

Journal: Theranostics

Article Title: Targeting ATF4-dependent pro-survival autophagy to synergize glutaminolysis inhibition

doi: 10.7150/thno.60028

Figure Lengend Snippet: ATF4 transcriptionally upregulates DDIT4 upon inhibition of glutaminolysis to inactivate mTOR. (A) Venn diagrams for the intersection of the candidate genes derived from ATF4 potential target genes, differentially expressed genes upon glutaminolysis inhibition, and hallmarks genes of mTORC1. (B) DDIT3, ASNS, DDIT4, NUPR1, PHGDH mRNA expression of cells treated by 968 for 24 h was detected by qRT-PCR. (C) DDIT4 expression of cells treated by 968 for 24 h was detected by western blotting. (D) The effect of DDIT4 knockdown on mTORC1 activity of cells treated by 968 for 24 h was detected by western blotting. p-mTOR, p-p70S6K, p-S6, DDIT4 band density was quantified and expressed as fold change, compared with the control, by arbitrarily setting the control value as 1. (E) The effect of DDIT4 knockdown on 968-induced cell apoptosis was measured by Flow Cytometry. The result presents the proportion of apoptotic cells. (F) The expression of cleaved PARP after DDIT4 knockdown and 968 treatment was detected by western blotting, cleaved PARP band density was quantified and expressed as fold change, compared with the control, by arbitrarily setting the control value as 1. (G) ATF4 enrichment at the DDIT4 promoter was determined with ChIP assay. (H) Schematic representation of ATF4 and dominant-negative ATF4ΔN, an ATF4 mutant lacks the N-terminal transcriptional activation domain was shown. The transcription activation ability of ATF4 and dominant-negative ATF4ΔN on the DDIT4 promoter was measured with luciferase assay. (I) The change of ATF4 enrichment at the DDIT4 promoter after 968 treatment was determined with ChIP assay. (J) DDIT4 protein expression after ATF4 knockdown during 968 treatment was detected by western blotting. ATF4, DDIT4 band density was quantified and expressed as fold change, compared with the control, by arbitrarily setting the control value as 1. (K) DDIT4 mRNA expression after ATF4 knockdown during 968 treatment was detected by qRT-PCR. Data are shown as the means ± SD from three experiments. For all experiments, statistical significance was assessed by Student's t -tests, * P < 0.05.

Article Snippet: DDIT4 (rabbit, 10638-1-AP) and YTHDF2 (rabbit, 24744-1-AP) polyclonal antibodies were purchased from Proteintech.

Techniques: Inhibition, Derivative Assay, Expressing, Quantitative RT-PCR, Western Blot, Knockdown, Activity Assay, Control, Flow Cytometry, Dominant Negative Mutation, Mutagenesis, Activation Assay, Luciferase

Journal: Theranostics

Article Title: Targeting ATF4-dependent pro-survival autophagy to synergize glutaminolysis inhibition

doi: 10.7150/thno.60028

Figure Lengend Snippet: Targeting ATF4-dependent pro-survival autophagy to synergize glutaminolysis inhibition. (A-B) Timeline showing that mice were treated with AOM and with 2% DSS as indicated. From day 30 on, the mice began to be treated with respective drugs until sacrifice. On day 100, mice were euthanized and the tissue was collected as shown in (B). (C) Numbers of tumors per mouse were counted (n = at least 4, * P < 0.05). (D) Tumor size in mm 3 /tumor were measured (n = at least 4, * P < 0.05). (E) Body weights of mice were recorded (n = at least 4, * P < 0.05). (F) IHC analysis of ATF4 expression in normal tissue, DMSO and 968 treated tumors tissue. (G) Working model: during glutaminolysis inhibition, FTO enhances ATF4 expression by reducing YTHDF2-mediated mRNA decay. ATF4 transcriptionally upregulates DDIT4 to inhibit mTORC1 activity and promote pro-survival autophagy. Finally, targeting the ATF4 pathway by autophagy inhibition combined with asparagine inhibitor is synthetic lethality with glutaminolysis inhibitor in CRC. Data are shown as the means ± SD (C, D, E). For all experiments, statistical significance was assessed by Student's t -tests, * P < 0.05. Scale bars are 100 μm in (F).

Article Snippet: DDIT4 (rabbit, 10638-1-AP) and YTHDF2 (rabbit, 24744-1-AP) polyclonal antibodies were purchased from Proteintech.

Techniques: Inhibition, Expressing, Activity Assay

Journal: Nature Communications

Article Title: piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis

doi: 10.1038/s41467-024-46664-3

Figure Lengend Snippet: a A schematic model showing the domain composition of MIWI and trajectory of the 5′ and 3′ ends of piRNA anchored with MID and PAZ-domain, respectively. The Y569/K573 and Y346/Y347 conserved in PIWI proteins are required for the 5′ end or 3′ end piRNA loading capacity of MIWI. b RNA co-IP assay of MIWI-associated-piRNAs (top) in wild-type (lane 1), Miwi YY/YY (lane 2), Miwi YK/YK (lane 3), and Miwi −/− testes (lane 4), with anti-MIWI IB as a loading reference (bottom). c , d Immunostaining of MIWI ( c , red) and TDRKH ( d , red) on testis sections from adult wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi −/ − mice using regular microscopy. Left: representative staining images of indicated mouse testis sections, scale bar, 20 μm; right, the enlargement of yellow framed regions, scale bar, 5 μm. The developmental stages of spermatocytes and spermatids were distinguished according to γH2AX (green) and DAPI (greyscale) staining. White arrows indicated MIWI ( c ) or TDRKH ( d ) aggregations. PS pachytene spermatocytes, DS diplotene spermatocytes, RS round spermatids. e Co-immunostaining of MIWI (red) and TDRKH (green) on testis sections from adult wildtype, Miwi YY/YY and Miwi YK/YK mice using confocal microscopy, with nuclei counterstained by DAPI (greyscale). White arrowheads indicated colocalization sites; yellow and white open arrowheads respectively indicated the unique localization of MIWI and TDRKH at non-colocalization sites. Scale bar, 10 μm. The results shown are representative of three independent experiments. Source data are provided as a Source Data file.

Article Snippet: Moreover, we serendipitously discovered that a commercially available rabbit monoclonal anti-MIWI antibody (ABclonal, Catalog No.: A3490, whose specificity was confirmed through immunoblotting of either epitope-deleted MIWI Δaa701-801 mutant, or MIWI protein in wildtype or Miwi −/− mouse testes; Fig. S ) selectively immunoprecipitated piRNA-unloaded MIWI protein in mouse testes as well as in transfected HEK293T cells (Figs and S ).

Techniques: Co-Immunoprecipitation Assay, Immunostaining, Microscopy, Staining, Confocal Microscopy

Journal: Nature Communications

Article Title: piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis

doi: 10.1038/s41467-024-46664-3

Figure Lengend Snippet: a , b Co-IP assay of the association of MIWI and TDRKH in mouse testes from 18 dpp wildtype, Miwi YY/YY , and Miwi YK/YK mice. Quantification of blot intensity of TDRKH protein in anti-MIWI pellets is shown in parentheses [the one from wildtype control mouse (lane 4) is set as 1.0 after normalization with MIWI blotting]. c RNA co-IP assay of MIWI-interacting piRNAs in anti-MIWI (lane 1) and anti-TDRKH IP pellets (lane 2) from adult wildtype mouse testes, with anti-MIWI IB as a loading reference (bottom). d Anti-MIWI unloaded preferably pulled down piRNA-unloaded MIWI (left) and TDRKH (right) in adult mouse testicular lysate. Left, RNA co-IP assays using anti-MIWI unloaded and control anti-MIWI antibodies in adult wildtype mouse testicular lysate, with anti-MIWI IB as loading references. Right, co-IP assay of the association of MIWI and TDRKH using anti-MIWI unloaded (lane 3) and control anti-MIWI antibodies (lane 4) in adult wildtype mouse testicular lysate, with testicular lysate (lane 1) and IgG IP (lane 2) serving as positive and negative controls, respectively. Quantification of blot intensity of TDRKH is shown in parentheses [the one anti-MIWI IP (lane 4) is set as 1.0 after normalization with MIWI blotting]. e RNase A treatment enhanced the MIWI-TDRKH interaction in wild-type mouse testes. Quantification of blot intensity of TDRKH protein in anti-MIWI pellets is shown in parentheses [the one from RNase A-untreated (lane 4) is set as 1.0 after normalization with MIWI blotting]. f Transfection of piRNA attenuated the MIWI-TDRKH interaction in Flag-tagged MIWI-stable-expressed GC-2spd (ts) cells. OE overexpression. Quantification of blot intensity of TDRKH is shown in parentheses [the one with RNase A-untreated and piRNA-free condition in Flag-tagged MIWI-stable-expressed GC-2spd (ts) cell lysates (lane 2) is set as 1.0 after normalization with MIWI blotting]. g Schematic diagram showing that piRNA loading facilitates the dissociation of MIWI from TDRKH. The results shown are representative of three independent experiments. Quantification of western blot analysis are represented as mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Moreover, we serendipitously discovered that a commercially available rabbit monoclonal anti-MIWI antibody (ABclonal, Catalog No.: A3490, whose specificity was confirmed through immunoblotting of either epitope-deleted MIWI Δaa701-801 mutant, or MIWI protein in wildtype or Miwi −/− mouse testes; Fig. S ) selectively immunoprecipitated piRNA-unloaded MIWI protein in mouse testes as well as in transfected HEK293T cells (Figs and S ).

Techniques: Co-Immunoprecipitation Assay, Control, Transfection, Over Expression, Western Blot

Journal: Nature Communications

Article Title: piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis

doi: 10.1038/s41467-024-46664-3

Figure Lengend Snippet: a Co-immunostaining of TDRKH (red) and TDRD6 (green) on testis sections from adult wildtype mice using confocal microscopy, with nuclei counterstained by DAPI (greyscale). PS pachytene spermatocytes, DS diplotene spermatocytes, RS round spermatids. Scale bar, 10 μm. b Co-immunostaining of MIWI (red) and TDRKH (green) on testis sections from adult wildtype and Tdrd6 − /− mice using confocal microscopy, with nuclei counterstained by DAPI (greyscale). White arrowheads indicated colocalization sites; yellow and white open arrowheads respectively indicated the unique localization of MIWI and TDRKH at non-colocalization sites. Scale bar, 10 μm. c , d Co-IP assay of the association of MIWI with TDRD6 and TDRKH in mouse testes from adult wildtype ( c and d , lanes 3 and 4), Miwi YY/YY ( c , lanes 5 and 6) and Miwi YK/YK ( d , lanes 5 and 6) mice. Anti-MIWI IP pellets ( c and d , lanes 4 and 6) were immunoblotted by the indicated antibodies, with testicular lysate ( c and d , lanes 1 and 2) and IgG IP ( c and d , lanes 3 and 5) serving as positive and negative controls, respectively. Quantification of blot intensity of indicated proteins in anti-MIWI IP pellets is shown in parentheses [the one from wildtype control mouse (lane 4) is set as 1.0 after normalization with MIWI blotting]. e Co-immunostaining of MIWI (red) and TDRD6 (green) on testis sections from adult wildtype, Miwi YY/YY and Miwi YK/YK mice using confocal microscopy, with nuclei counterstained by DAPI (greyscale). White arrowheads indicated colocalization sites. Scale bar, 10 μm. The results shown are representative of three independent experiments. Quantification of western blot analysis are represented as mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Moreover, we serendipitously discovered that a commercially available rabbit monoclonal anti-MIWI antibody (ABclonal, Catalog No.: A3490, whose specificity was confirmed through immunoblotting of either epitope-deleted MIWI Δaa701-801 mutant, or MIWI protein in wildtype or Miwi −/− mouse testes; Fig. S ) selectively immunoprecipitated piRNA-unloaded MIWI protein in mouse testes as well as in transfected HEK293T cells (Figs and S ).

Techniques: Immunostaining, Confocal Microscopy, Co-Immunoprecipitation Assay, Control, Western Blot

Journal: Nature Communications

Article Title: piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis

doi: 10.1038/s41467-024-46664-3

Figure Lengend Snippet: a RNase A treatment barely altered MIWI methylation and MIWI-TDRD6 interaction in the adult testicular lysate. Quantification is shown in parentheses [the one from RNase A-untreated (lane 2) is set as 1.0 after normalization with MIWI blotting]. b Co-IP assay of the association of MIWI (lane 2) or arginine methylation-deficient MIWI R-K mutant (lane 3) with TDRD6 in co-transfected HEK293T cells. Quantification is shown in parentheses [the one with wildtype MIWI (lane 2) is set as 1.0 after normalization with MIWI blotting]. c Co-IP assay of the effect of methyltransferase inhibitor methylthioadenosine (MTA, Sigma, D5011) on the MIWI-TDRD6 interaction in co-transfected HEK293T cells. Quantification is shown in parentheses [the one with DMSO treatment (lane 1) is set as 1.0 after normalization with MIWI blotting]. d , e piRNA loading-deficient mutations impaired arginine methylation of MIWI in mouse testes. Quantification is shown in parentheses [the one from the wildtype control mouse (lane 2) is set as 1.0 after normalization with MIWI blotting]. f Anti-MIWI unloaded antibody pulled down less methylated MIWI and TDRD6 in adult wildtype mouse testicular lysate (lane 3) compared with control anti-MIWI antibody (lane 4). Quantification is shown in parentheses [the one anti-MIWI IP (lane 4) is set as 1.0 after normalization with MIWI blotting]. g Sequential co-IP showing that TDRKH is mainly associated with unmethylated MIWI. Quantification is shown in parentheses [the first anti-MIWI IP (lane 2) is set as 1.0 after normalization with MIWI blotting]. h TDRKH reduced MIWI methylation in co-transfected HEK293T cells. Quantification is shown in parentheses [the one without TDRKH transfection (lane 1) is set as 1.0 after normalization with MIWI blotting]. i Schematic diagram showing that piRNA loading promotes MIWI dissociation from TDRKH, leading to the exposure of the N-terminal of MIWI for arginine methylation by PRMT5 to enhance the MIWI-TDRD6 interaction. The results shown are representative of three independent experiments. Quantification of western blot analysis are represented as mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Moreover, we serendipitously discovered that a commercially available rabbit monoclonal anti-MIWI antibody (ABclonal, Catalog No.: A3490, whose specificity was confirmed through immunoblotting of either epitope-deleted MIWI Δaa701-801 mutant, or MIWI protein in wildtype or Miwi −/− mouse testes; Fig. S ) selectively immunoprecipitated piRNA-unloaded MIWI protein in mouse testes as well as in transfected HEK293T cells (Figs and S ).

Techniques: Methylation, Co-Immunoprecipitation Assay, Mutagenesis, Transfection, Control, Western Blot

Journal: Nature Communications

Article Title: piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis

doi: 10.1038/s41467-024-46664-3

Figure Lengend Snippet: a All tested Miwi YY/YY and Miwi YK/YK males were infertile. b Testes from adult Miwi YY/YY and Miwi YK/YK mice were moderately reduced compared with wildtype control. Left, a representative image of testes from indicated mice; right, the average weight of testes from wildtype, Miwi YY/YY ( p = 0.033) and Miwi YK/YK ( p = 0.014) mice ( n = 6, data are represented as mean ± SD, P values were calculated using two-tailed Student’s t -test, *p < 0.05). c PAS staining of the testis (top) and H&E staining of the epididymis (bottom) sections from adult wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi −/− mice. Scale bar, 30 μm. d Acrosome staining (ACRV1, red) of testis sections from wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi − /− mice using regular microscopy. Developmental stages of the seminiferous tubules were distinguished according to γH2AX (green) and DAPI (grayscale) staining. Scale bar, 10 μm. e TUNEL assays (red) of testis sections from adult wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi −/ − mice using regular microscopy, with nuclei counterstained by DAPI (blue). Scale bar, 30 μm. Results shown in c – e are representative of three independent experiments. Source data are provided as a Source Data file.

Article Snippet: Moreover, we serendipitously discovered that a commercially available rabbit monoclonal anti-MIWI antibody (ABclonal, Catalog No.: A3490, whose specificity was confirmed through immunoblotting of either epitope-deleted MIWI Δaa701-801 mutant, or MIWI protein in wildtype or Miwi −/− mouse testes; Fig. S ) selectively immunoprecipitated piRNA-unloaded MIWI protein in mouse testes as well as in transfected HEK293T cells (Figs and S ).

Techniques: Control, Two Tailed Test, Staining, Microscopy, TUNEL Assay

Journal: Nature Communications

Article Title: piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis

doi: 10.1038/s41467-024-46664-3

Figure Lengend Snippet: a Detection of piRNA expression in adult wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi −/− testes. b The length distribution of small RNAs from adult wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi −/− testes. Data were normalized by miRNA reads (21–23 nt). c Nucleotide distributions at the first position in the piRNAs from adult wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi −/− testes. d Genomic annotation of the piRNAs from adult wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi −/− testes. The percentage of mapped reads is shown. e Scatter plot of total piRNA reads mapped to individual piRNA clusters from adult wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi −/− testes. Data were normalized by miRNA reads (21–23 nt). f Western blotting of MIWI and MILI expression in testes from wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi −/− mice with indicated ages. β-actin served as a loading control. Quantification of blot intensity of MIWI is shown in parentheses (the one in wildtype testis is set as 1.0 after normalization with β-actin). Results shown in a and f are representative of three independent experiments, and small RNA-seq experiments shown in b – e with two replicates. Quantification of western blot analysis are represented as mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Moreover, we serendipitously discovered that a commercially available rabbit monoclonal anti-MIWI antibody (ABclonal, Catalog No.: A3490, whose specificity was confirmed through immunoblotting of either epitope-deleted MIWI Δaa701-801 mutant, or MIWI protein in wildtype or Miwi −/− mouse testes; Fig. S ) selectively immunoprecipitated piRNA-unloaded MIWI protein in mouse testes as well as in transfected HEK293T cells (Figs and S ).

Techniques: Expressing, Western Blot, Control, RNA Sequencing

Journal: Nature Communications

Article Title: piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis

doi: 10.1038/s41467-024-46664-3

Figure Lengend Snippet: Upon its expression in mid-pachytene spermatocytes, MIWI protein is recruited to the IMC for piRNA processing via interacting with TDRKH through its unmethylated N-terminus, while piRNA loading induces a conformational change of MIWI and, in turn, weakens its interaction with TDRKH, leading to its release from the IMC. Meanwhile, the disassociation of MIWI with TDRKH simultaneously results in the arginine residues in its N-terminus exposed for methylation by PRMT5, thereby enhancing TDRD6 binding to prime its localization in the CB for piRNA function.

Article Snippet: Moreover, we serendipitously discovered that a commercially available rabbit monoclonal anti-MIWI antibody (ABclonal, Catalog No.: A3490, whose specificity was confirmed through immunoblotting of either epitope-deleted MIWI Δaa701-801 mutant, or MIWI protein in wildtype or Miwi −/− mouse testes; Fig. S ) selectively immunoprecipitated piRNA-unloaded MIWI protein in mouse testes as well as in transfected HEK293T cells (Figs and S ).

Techniques: Expressing, Methylation, Binding Assay

Journal: Nature Communications

Article Title: AMPK induces degradation of the transcriptional repressor PROX1 impairing branched amino acid metabolism and tumourigenesis

doi: 10.1038/s41467-022-34747-y

Figure Lengend Snippet: a , b TMT6 labelled quantitative proteomics ( a ) to investigate the mechanisms for tumour cell metabolic plasticity and KEGG pathway enrichment analysis ( b ) the differential protein in the Huh7 cells upon glucose starvation (Glc starv.) for 12 h. c Volcano plot shows the total proteins upon glucose starvation by LC-MS/MS. d Comparison of the PROX1 mRNA level between KRAS/TP53/LKB1 wild-type (WT), single KRAS mutation (KRAS), KRAS/TP53 both mutation and KRAS/LKB1both mutation in the lung adenocarcinoma (LUAD) from TCGA database. WT/KRAS/KP/KL: n = 138/66/47/31, maximum = 11.54/12.69/11.74/11.57, upper quartile = 8.95/9.11/8.76/10.28, median = 8.06/8.36/8.10/9.32, lower quartile = 7.63/7.64/7.47/8.17, minimum = 6.16/6.75/6.31/7.24. e , f Western blot analysis the cell lysates upon glucose starvation ( e ) and metformin treatment ( f ) as indicated. g Immunofluorescence analysis the Huh7 cells as indicated ( n = 3 independent experiments). Scale bar, 10 µm. h , i Liver tissues from normal, fasted and metformin (500 mg/L) treatment mice ( n = 6) are subjected to immunohistochemistry ( h ) and the corresponding quantified graph of liver tissues ( i ) are shown. Scale bar, 50 µm. j , k The apoptotic analysis of the Huh7 ( j ) and HepG2 ( k ) cells were infected with the lentivirus either expressing PROX1 siRNA (si259 or si1646) precursor or scrambled siRNA precursor (SCR) by flow cytometry ( n = 3 independent experiments). l The apoptotic analysis of the wild-type (WT) and AMPKα knockout (Ampkα -/- ) MEFs were infected as indicated ( n = 3). m Immunoblot analysis the cell lysates as indicated. n Western blot analysis the cell lysates overexpression of the wild-type AMPKα2 (WT-AMPK), the constitutively active AMPK (CA-AMPK) and the dominant-negative AMPK (Dn-AMPK). o Representative confocal images of Huh7 cells transfected with WT-, CA- and Dn-AMPKα2 plasmids ( n = 3 independent experiments). Scale bar, 10 µm. p Western blot analysis the cell lysates as indicated. q , r Representative IHC staining images ( q ) and statistical data ( r ) in the murine lung tumour tissues from Kras G12D / Lkb1 L/L and Kras G12D -sgAmpk mouse ( n = 5). Scale bar, 50 µm. The immunoblots are repeated independently with similar results at three times. For i–l and r , data represent the mean ± SD. Statistical significance was assessed using two-tailed unpaired Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against the following proteins were obtained from Cell Signaling Technology: p-AMPK (#2535, 1:200); from Abcam: DDB1 (ab109027, 1:200); from Abclonal: p-S79 (customization, 1:100), ACADM (A4567, 1:200), EHHADH (A13488, 1:200) and HIBADH (A19871, 1:200); from Proteintech: PROX1 (11067-1-AP, 1:200), CUL4A (14851-1-AP, 1:200), CUL4B (12916-1-AP, 1:200), BCAT1 (13640-1-AP, 1:200), BCKDHB (13685-1-AP, 1:200), ACADSB (13122-1-AP, 1:200), DLD (16431-1-AP, 1:200) and HMGCL (16898-1-AP, 1:200).

Techniques: Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Western Blot, Immunofluorescence, Immunohistochemistry, Infection, Expressing, Flow Cytometry, Knock-Out, Over Expression, Dominant Negative Mutation, Transfection, Two Tailed Test

Journal: Nature Communications

Article Title: AMPK induces degradation of the transcriptional repressor PROX1 impairing branched amino acid metabolism and tumourigenesis

doi: 10.1038/s41467-022-34747-y

Figure Lengend Snippet: a The Ser79 phosphorylation modification of PROX1 peptides identified through liquid chromatography-tandem mass spectrometry. b The substrate motif of AMPK kinases is shown (lower), and the Ser79 site of PROX1 is conserved in vertebrate. c Coomassie blue staining of GST and GST-AMPKα2 incubated with in vitro translated PROX1, PROX1 was detected by anti-PROX1 antibody after GST-pulldown. d Endogenous PROX1 and AMPKα2 in the Huh7 cells were visualized under fluorescent microscopy ( n = 3 independent experiments). Nuclei were stained with DAPI. Scale bar, 10 µm. e Western blot analysis Huh7 cell lysates as indicated. f The domain organization of PROX1 and the deletion constructs. PD1, prospero domain1; HD, homeodomain; PD2, prospero domain 2. g Input, coomassie blue staining of each GST-PROX1 fragment incubated with in vitro translated AMPKα2. AMPKα2 was detected by anti-AMPKα2 antibody after GST-pulldown. h The P1-WT and P1-S79A of PROX1 as indicated was detected using a phosphor-specific antibody against Ser79 of PROX1. i HEK293T-expressed wide type (WT) and replacement of S79 with Ala (A) or Glu (E) in the FLAG-PROX1 (S79A and S79E) incubated with GST-AMPKα2. j Western blot analysis HEK293T cell lysates as indicated. k Immunoblot analysis of HEK293T cell lysates transfected with the indicated PROX1 and HA-AMPKα2 plasmids. l Immunoblot analysis of the FLAG-IP and cell lysates from transfected with the indicated constructs. m Immunoblot analysis of MEFs cell lysates as indicated. n Representative IHC staining images and statistical data of the murine lung tumour tissues from Kras G12D (K), Kras G12D / Lkb1 L/L (KL) and Kras G12D -sgAmpk (KA) mouse ( n = 5). Scale bar, 50 µm. o Representative IHC staining images and statistical data of the liver tissues from normal, fasted and metformin (500 mg/L) treatment mice ( n = 6). Scale bar, 50 µm. p, q Representative western blot ( p ) and the corresponding quantified graph ( q ) of Huh7 cell lysates are shown. n = 3 independent experiments. IB, immunoblot; IP, immunoprecipitation. The immunoblots are repeated independently with similar results at three times. For n, o and q , data represent the mean ± SD. Statistical significance was assessed using two-tailed unpaired Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against the following proteins were obtained from Cell Signaling Technology: p-AMPK (#2535, 1:200); from Abcam: DDB1 (ab109027, 1:200); from Abclonal: p-S79 (customization, 1:100), ACADM (A4567, 1:200), EHHADH (A13488, 1:200) and HIBADH (A19871, 1:200); from Proteintech: PROX1 (11067-1-AP, 1:200), CUL4A (14851-1-AP, 1:200), CUL4B (12916-1-AP, 1:200), BCAT1 (13640-1-AP, 1:200), BCKDHB (13685-1-AP, 1:200), ACADSB (13122-1-AP, 1:200), DLD (16431-1-AP, 1:200) and HMGCL (16898-1-AP, 1:200).

Techniques: Modification, Liquid Chromatography, Mass Spectrometry, Staining, Incubation, In Vitro, Microscopy, Western Blot, Construct, Transfection, Immunohistochemistry, Immunoprecipitation, Two Tailed Test

Journal: Nature Communications

Article Title: AMPK induces degradation of the transcriptional repressor PROX1 impairing branched amino acid metabolism and tumourigenesis

doi: 10.1038/s41467-022-34747-y

Figure Lengend Snippet: a Interaction between PROX1 and CUL proteins were analyzed in the HEK293T cells. IP, immunoprecipitation. b Immunoblot analysis of the cell lysates from Huh7 cells transfected with the indicated dominant-negative CUL (dn-CUL1, 2, 3, 4 A, 4B and 5) constructs. c Endogenous PROX1 in the Huh7 cells treated with MG132 (4uM) was immunoprecipitated using anti-PROX1 antibody or isotype IgG control and subjected to immunoblot analysis. d HepG2 cells transfected with FLAG-PROX1 were deprived of glucose for 8 h in the presence of MG132. e Representative confocal images from Huh7 cells ( n = 3 independent experiments). Scale bar, 10 µm. f Purified recombinant GST-PROX1 (P1-WT, S79A and S79E) was incubated with DDB1 individually. g Representative confocal images of PROX1 expression in the Huh7 cells transfected with WT- and Dn-CUL4A, 4B constructs, and Flag-DDB1 as the indicated ( n = 3 independent experiments). Scale bar, 20 µm. h , i HepG2 cells infected with shRNA lentivirus encoding scramble (SCR) and shCUL4B were treated cycloheximide (CHX, 100 mg/mL). Representative western blot ( h ) and the corresponding quantified graph ( i ) are shown ( n = 3 independent experiments). j , k HepG2 cells infected with the lentivirus as indicated. Representative western blot ( j ) and the corresponding quantified graph ( k ) are shown ( n = 3 independent experiments). l, m HEK293T cells transfected with FLAG-PROX1 variants upon glucose starvation were subject to CHX (100 mg/mL) treatment. Representative western blot ( l ) and the corresponding quantified graph ( m ) are shown ( n = 3 independent experiments). n Ubiquitination levels of FLAG-PROX1 variants in the HEK293T cells upon glucose starvation were immunoprecipitated using anti-FLAG mAb and subjected to immunoblot analysis. o Immunoblot analysis of the cell lysates from HEK293T cells co-transfected plasmids as indicated. p , q Representative IHC staining images ( p ) and the heatmap ( q ) of IHC score (by Pearson’s) between PROX1, CUL4A, CUL4B and DDB1 expression in HCC tissues ( n = 90). Scale bar, 50 µm. The immunoblots are repeated independently with similar results at three times. For i, k and m , data represent the mean ± SD. Statistical significance was assessed using two-tailed unpaired Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against the following proteins were obtained from Cell Signaling Technology: p-AMPK (#2535, 1:200); from Abcam: DDB1 (ab109027, 1:200); from Abclonal: p-S79 (customization, 1:100), ACADM (A4567, 1:200), EHHADH (A13488, 1:200) and HIBADH (A19871, 1:200); from Proteintech: PROX1 (11067-1-AP, 1:200), CUL4A (14851-1-AP, 1:200), CUL4B (12916-1-AP, 1:200), BCAT1 (13640-1-AP, 1:200), BCKDHB (13685-1-AP, 1:200), ACADSB (13122-1-AP, 1:200), DLD (16431-1-AP, 1:200) and HMGCL (16898-1-AP, 1:200).

Techniques: Immunoprecipitation, Western Blot, Transfection, Dominant Negative Mutation, Construct, Purification, Recombinant, Incubation, Expressing, Infection, shRNA, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: AMPK induces degradation of the transcriptional repressor PROX1 impairing branched amino acid metabolism and tumourigenesis

doi: 10.1038/s41467-022-34747-y

Figure Lengend Snippet: a Venn diagram showing the number genes in the mouse liver with PROX1 binding and displaying expression changes in Prox1 liver specific knockout mice (Alb-Cre; Prox1 f/f ). KEGG pathway enrichment analysis the above overlapping genes as indicated. b Heatmap demonstration of the gene expression related to valine, leucine and isoleucine degradation (BCAA metabolism) from WT and Prox1-cKO mice ( n = 3). c GSEA shows the enrichment of BCAA metabolism in Alb-Cre; Prox1 f/f mice. d GSEA shows the enrichment of BCAA metabolism in Kras G12D mice compared with the Kras G12D / Lkb1 L/L mice. Statistical significance was assessed using Permutation test. e Real-time PCR analysis the relative mRNA levels of BCAA metabolism genes from Prox1 f/f and Alb-Cre; Prox1 f/f mice ( n = 3). f Real-time PCR analysis the relative mRNA levels from Huh7 cells as indicated ( n = 3). g Real-time PCR analysis the relative mRNA levels of Huh7 cells as indicated ( n = 3). h , i ChIP-qrtPCR was performed with sonicated chromatins immunoprecipitated from Huh7 cells ( h ) and mouse liver tissue ( i ) by anti-PROX1 antibody or preimmune IgG ( n = 3). j Heatmap of the gene peaks by ATAC-seq from WT and Prox1-cKO mouse liver tissues. k Density maps for ATAC-seq in liver tissues from WT and Prox1-cKO mice. l , m ChIP analysis of H3K4me3 ( l ) and H3K9me3 ( m ) enrichment in mouse liver tissues as indicated ( n = 3). n Real-time PCR analysis the relative mRNA levels as indicated ( n = 3). o ChIP analysis of H3K9me3 enrichment at the BCAA embolism genes promoter as indicated ( n = 3). p Immunoblot analysis cell lysates from Huh7 cells and liver tissues from WT and Alb-Cre; Prox1 f/f (cKO) mice as indicated ( n = 3). q , r Representative IHC staining images ( q ) and the heatmap of IHC score (by Pearson’s) ( r ) between PROX1 and several proteins as indicated in HCC tissues ( n = 90). Scale bar, 50 µm. The immunoblots are repeated independently with similar results at three times. n was biological replicates for all experiments. For e–i and l–o , data represent the mean ± SD and * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical significance was assessed using two-tailed unpaired Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against the following proteins were obtained from Cell Signaling Technology: p-AMPK (#2535, 1:200); from Abcam: DDB1 (ab109027, 1:200); from Abclonal: p-S79 (customization, 1:100), ACADM (A4567, 1:200), EHHADH (A13488, 1:200) and HIBADH (A19871, 1:200); from Proteintech: PROX1 (11067-1-AP, 1:200), CUL4A (14851-1-AP, 1:200), CUL4B (12916-1-AP, 1:200), BCAT1 (13640-1-AP, 1:200), BCKDHB (13685-1-AP, 1:200), ACADSB (13122-1-AP, 1:200), DLD (16431-1-AP, 1:200) and HMGCL (16898-1-AP, 1:200).

Techniques: Binding Assay, Expressing, Knock-Out, Real-time Polymerase Chain Reaction, Sonication, Immunoprecipitation, Western Blot, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: AMPK induces degradation of the transcriptional repressor PROX1 impairing branched amino acid metabolism and tumourigenesis

doi: 10.1038/s41467-022-34747-y

Figure Lengend Snippet: a Relative abundance of amino acids by LC/MS in liver tissues of Prox1 f/f (WT) and Alb-Cre; Prox1 f/f (Prox1-cKO) mice ( n = 6). b Relative abundance of valine, leucine and isoleucine by LC/MS in Huh7 cells infected with the lentivirus either expressing PROX1 siRNA (si259 or si1646) precursor or scrambled siRNA precursor (SCR) ( n = 3). c Relative abundance of BCAA in the liver tissues as indicated ( n = 3). d Relative abundance of BCAA in the Huh7 cells as indicated ( n = 3). e , f Schematic of isotope tracing in Huh7 cells were traced 24 h with [ 15 N, 13 C]-Gln ( e ), followed by LC/MS analysis of the labelled metabolites ( f ) ( n = 4). g LC/MS analysis of the labelled metabolites in Huh7 cells upon glucose starvation were traced 24 h with [ 15 N, 13 C]-Gln ( n = 4). h , i Schematic of isotope tracing in Huh7 cells were traced 24 h with [ 13 C]-Leu ( h ), followed by LC/MS analysis of the labelled metabolites ( i ) ( n = 4). j LC/MS analysis of the labelled metabolites in Huh7 cells as indicated upon glucose starvation were traced 24 h with [ 13 C]-Leu ( n = 4). k GSEA shows the enrichment of mTOR pathway in the Prox1 f/f mice. Statistical significance was assessed using Permutation test. l Immunoblot analysis of the cell lysates as indicated. m Immunoblot analysis of Huh7 cells with or without Leu (200 µM) as indicated. n Immunoblot analysis of the lysates from the liver tissues with or without BCAA (200%) as indicated, and the relative p-S6K level are shown ( n = 3). o Representative IHC staining images (upper) and statistical data (down) of p-S6K and PROX1 expression in HCC tissues ( n = 90). Scale bar, 50 µm. p Immunoblot analysis of Huh7 cell lysates as indicated and the relative p-S6K level are shown ( n = 3). q Immunoblot analysis of SMMC-7721 cell lysates as indicated and the relative p-S6K level are shown ( n = 3). The immunoblots are repeated independently with similar results at three times. n was biological replicates for all experiments. For a–d, f–g, i–j, n and p–q , data represent the mean ± SD. Statistical significance was assessed using two-tailed unpaired Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against the following proteins were obtained from Cell Signaling Technology: p-AMPK (#2535, 1:200); from Abcam: DDB1 (ab109027, 1:200); from Abclonal: p-S79 (customization, 1:100), ACADM (A4567, 1:200), EHHADH (A13488, 1:200) and HIBADH (A19871, 1:200); from Proteintech: PROX1 (11067-1-AP, 1:200), CUL4A (14851-1-AP, 1:200), CUL4B (12916-1-AP, 1:200), BCAT1 (13640-1-AP, 1:200), BCKDHB (13685-1-AP, 1:200), ACADSB (13122-1-AP, 1:200), DLD (16431-1-AP, 1:200) and HMGCL (16898-1-AP, 1:200).

Techniques: Liquid Chromatography with Mass Spectroscopy, Infection, Expressing, Western Blot, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: AMPK induces degradation of the transcriptional repressor PROX1 impairing branched amino acid metabolism and tumourigenesis

doi: 10.1038/s41467-022-34747-y

Figure Lengend Snippet: a–c Analysis of Prox1 f/f (WT) and Alb-Cre; Prox1 f/f (Prox1-cKO) mice with DEN-induced liver cancer with normal or high BCAA (200%) diets. Representative images of livers ( a ) and the number of tumours ( b ), and the Liver/body weight ( c ) of the mice ( n = 5) as indicated. Scale bar, 1 cm. d–f Representative images and H&E staining of livers ( d ) and the number of tumours ( e ), and the Liver/body weight ( f ) of the mice ( n = 5) as indicated. g Huh7 cells stably expressing the indicated siRNAs were subcutaneously injected into in nude mice respectively with or without metformin (500 mg/L) treatment. Shown are average tumour volumes over time ( n = 7). Data are presented as mean ± SEM. h SMCC-7721 cells stably overexpressing the PROX1 variants were subcutaneously injected into nude mice respectively with or without metformin (500 mg/L) treatment as indicated, and the weights of tumours are shown ( n = 6). i Schematic model of lung tumourigenesis from Kras G12D / Lkb1 L/L mice treated with a lenti-Cre-PROX1 virus. Eight weeks after nasal inhalation, mice were killed and analysed. j–k Representative H&E image ( j ) of lung tumour are shown. Scale bar, 500 µm. The tumour burden ( k ) and average tumour numbers ( l ) were calculated and plotted ( n = 5 mice for each group). m Immunohistochemistry analysis of the levels of Prox1, p-S6K, Bckdhb, Acadsb and Ehhadh in the above tissues as indicated. Scale bar, 50 µm. n–p Representative the H&E image ( n ) of lung tumour from Kras G12D / Lkb1 L/L mice treated with a lenti-Cre-sgPROX1 and sgSCR virus as indicated with or without phenformin (1.5 g/L) treatment ( n = 5 mice for each group). Scale bar, 500 µm. Statistical analysis of the tumour burden ( o ) and average tumour numbers ( p ) were shown. q , r Representative IHC staining images ( q ) and statistical data ( r ) of Ki67 expression in the above tissues ( n = 5) as indicated. Scale bar, 50 µm. For b–c, e–f, h, k–l, o–p and r , data represent the mean ± SD. Statistical significance was assessed using two-tailed unpaired Student’s t -test. n.s. not significant. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against the following proteins were obtained from Cell Signaling Technology: p-AMPK (#2535, 1:200); from Abcam: DDB1 (ab109027, 1:200); from Abclonal: p-S79 (customization, 1:100), ACADM (A4567, 1:200), EHHADH (A13488, 1:200) and HIBADH (A19871, 1:200); from Proteintech: PROX1 (11067-1-AP, 1:200), CUL4A (14851-1-AP, 1:200), CUL4B (12916-1-AP, 1:200), BCAT1 (13640-1-AP, 1:200), BCKDHB (13685-1-AP, 1:200), ACADSB (13122-1-AP, 1:200), DLD (16431-1-AP, 1:200) and HMGCL (16898-1-AP, 1:200).

Techniques: Staining, Stable Transfection, Expressing, Injection, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: AMPK induces degradation of the transcriptional repressor PROX1 impairing branched amino acid metabolism and tumourigenesis

doi: 10.1038/s41467-022-34747-y

Figure Lengend Snippet: a , b Representative IHC staining images ( a ) and statistical data ( b ) of p-S79 and p-AMPK expression in HCC tissues ( n = 90). Scale bar, 500 µm. c Kaplan–Meier analysis of overall survival probability of p-S79 levels in HCC patients. The statistical significance was assessed using log-rank test according to HCC patients with low or high expression of p-S79. d Kaplan–Meier analysis of overall survival in the HCC patients ( n = 90) according to combined expression status of p-S79 and p-AMPK. e , f Representative IHC staining images ( e ) and statistical data ( f ) of p-S79 and p-AMPK expression in lung adenocarcinoma (LUAD) tissues ( n = 90). Scale bar, 500 µm. g , h Kaplan–Meier analysis of overall survival ( g ) and disease-free survival ( h ) probability of PROX1 levels in LUAD patients ( n = 90). i , j Kaplan–Meier analysis of overall survival ( i ) and disease-free survival ( j ) probability of p-S79 levels in LUAD patients. k , l Kaplan–Meier analysis of overall survival ( k ) and disease-free survival ( l ) in NSCLC patients according to combined expression status of p-S79 and p-AMPK. m Glucose deprivation activated AMPK directly phosphorylated PROX1 at Ser79, allowing a rapid recruitment of Cul4-DDB1 E3 ubiquitin ligase complex to promote PROX1 degradation, a critical event that activates BCAA metabolism to suppress mTOR signalling pathway. Conversely, the deficient-LKB1-AMPK axis in cancers reactivates PROX1 to dictate BCAA catabolism and mTOR signalling, facilitating tumourigenesis and aggressiveness. For b and f , statistical significance was assessed using two-sided Chi-square test. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against the following proteins were obtained from Cell Signaling Technology: p-AMPK (#2535, 1:200); from Abcam: DDB1 (ab109027, 1:200); from Abclonal: p-S79 (customization, 1:100), ACADM (A4567, 1:200), EHHADH (A13488, 1:200) and HIBADH (A19871, 1:200); from Proteintech: PROX1 (11067-1-AP, 1:200), CUL4A (14851-1-AP, 1:200), CUL4B (12916-1-AP, 1:200), BCAT1 (13640-1-AP, 1:200), BCKDHB (13685-1-AP, 1:200), ACADSB (13122-1-AP, 1:200), DLD (16431-1-AP, 1:200) and HMGCL (16898-1-AP, 1:200).

Techniques: Immunohistochemistry, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Small G Proteins Rac1 and Ras Regulate Serine/Threonine Protein Phosphatase 5 (PP5)·Extracellular Signal-Regulated Kinase (ERK) Complexes Involved in the Feedback Regulation of Raf1 *

doi: 10.1074/jbc.M113.518514

Figure Lengend Snippet: Analysis of PP5-ERK interactions and PP5 activity. A, HEK-293FT cells were transfected with FLAG-PP5, HA-ERK1, or HA-ERK2 alone or in combination, as indicated. Western analysis of FLAG immune complexes (FLAG IPs), HA immune complexes (HA IPs), and cell lysates were performed using FLAG, HA, and HSP90 antibodies. *, IgG heavy chain. B, HEK-293FT cells were transfected with FLAG-PP5, HA-ERK1b, or HA-ERK1c alone or in combination, as indicated. FLAG IPs and cell lysates were analyzed by Western as in A. C, HEK-293FT cells were transfected with wild type FLAG-PP5 or FLAG-PP5HBD alone or together with HA-ERK1 or HA-ERK2; cells were also transfected with the indicated HA-tagged kinase and pcDNA3 (Vector). FLAG IPs and cell lysates were analyzed as described in A. A significant reduction in the binding of HA-ERK1 (88.21 ± 3.01%, p < 0.0001) and HA-ERK2 (77.3 ± 8.97%, p = 0.0003) to FLAG-PP5HBD was found when ERK signals were normalized to levels of mutant PP5 in the IPs and compared with the corresponding values in the wild type FLAG-PP5 conditions, which were set to 100. The results represent the means ± S.E. analyzed by one-sample t test using two-tailed p values. D, approximately 118 ng (10 nm) of purified wild type FLAG-PP5 (WT) or HSP90 binding-deficient mutant of FLAG-PP5 (HBD) were continuously assayed over 900 s for phosphatase activity toward DiFMUP (relative fluorescent units (RFU)) in the presence of only buffer (Basal), 100 μm arachidonic acid (AA), or 200 nm S100B plus 1 mm CaCl2 (S100B). Background fluorescence (i.e. samples containing only DiFMUP + arachidonic acid or DiFMUP + S100B + CaCl2) was measured and subtracted from the corresponding fluorescent values of the phosphatase-containing samples. Levels of fluorescence in WT + arachidonic acid, HBD + arachidonic acid, and HBD + S100B preparations were virtually identical. The results represent the means ± S.E. from six independent experiments, three experiments performed with duplicates from each of two separate purifications of WT and HBD. S.E. bars are obscured by the symbols for most data points. E, quantification of phosphatase activity at the 900 s time point. Two-way analysis of variance identified a statistically significant genotype versus activator interaction (F(5,30) = 61.83, p < 0.0001). Tukey post-tests are shown as follows: ***, versus basal, p < 0.0001; ^^^, WT versus HBD, p < 0.0001. Error bars, S.E. F, HEK-293FT cells expressing FLAG-PP5 and HA-ERK2 were lysed in Buffer B (B) or RIPA buffer (R) (Lysates). FLAG immunoprecipitations (FLAG IPs) were performed from the cell lysates and washed (IP Wash) in either Buffer B or RIPA buffer, as indicated. The FLAG IPs and corresponding cell lysates were analyzed by Western using HSP90, HA, and FLAG antibodies. The data are representative of experiments performed three (A), three (B), six (C), six (D), and two (F) independent times with similar results.

Article Snippet: The rabbit anti-PP5 antibody was from Bethyl Laboratories, Inc. (Montgomery, TX).

Techniques: Activity Assay, Transfection, Western Blot, Plasmid Preparation, Binding Assay, Mutagenesis, Two Tailed Test, Purification, Fluorescence, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Small G Proteins Rac1 and Ras Regulate Serine/Threonine Protein Phosphatase 5 (PP5)·Extracellular Signal-Regulated Kinase (ERK) Complexes Involved in the Feedback Regulation of Raf1 *

doi: 10.1074/jbc.M113.518514

Figure Lengend Snippet: The interaction of PP5 with ERK1/2 is independent of kinase and phosphatase activity. A, FLAG immunoprecipitations (FLAG IPs) were performed from lysates of HEK-293FT cells transfected with wild type FLAG-PP5, kinase-dead ERK1 (HA-ERK1KD), or kinase-dead ERK2 (HA-ERK2KD) alone or in combination, as indicated. The cell lysates and FLAG IPs were subjected to Western analysis using FLAG, HA, and HSP90 antibodies. B, FLAG IPs were performed from lysates of HEK-293FT cells transfected with phosphatase-dead PP5 (FLAG-PP5PD), wild type HA-ERK1, or wild type HA-ERK2 alone or in combination, as indicated. The cell lysates and FLAG IPs were analyzed as described in A. *, IgG heavy chain. The data are representative of experiments performed three (A) and three (B) independent times with similar results.

Article Snippet: The rabbit anti-PP5 antibody was from Bethyl Laboratories, Inc. (Montgomery, TX).

Techniques: Activity Assay, Transfection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Small G Proteins Rac1 and Ras Regulate Serine/Threonine Protein Phosphatase 5 (PP5)·Extracellular Signal-Regulated Kinase (ERK) Complexes Involved in the Feedback Regulation of Raf1 *

doi: 10.1074/jbc.M113.518514

Figure Lengend Snippet: Active Rac1 promotes assembly of PP5·ERK1 and PP5·ERK2 complexes. A, HEK-293FT cells were co-transfected with FLAG-PP5 and either HA-ERK1 or HA-ERK2 together with pcDNA3 (Vector), constitutively active Rac1 (Rac1L61), or dominant negative Rac1 (Myc-Rac1N17), as indicated. Western analysis of FLAG immune complexes (FLAG IPs) and cell lysates were performed using the HA, FLAG, HSP90, and Rac1 antibodies. B, quantification of the percentage of maximal HA-ERK binding normalized to the FLAG-PP5 signal in FLAG IPs, with binding in the vector samples set to 100. One-way analysis of variance identified a significant increase in PP5-ERK1 (F(2,6) = 6.222, p = 0.0344) and PP5-ERK2 (F(2,6) = 17.28, p = 0.0032) association in the presence of Rac1L61. Tukey post-tests are shown as follows: *, p < 0.05; **, p < 0.01. Data are mean ± S.E. No significant differences in the expression levels of FLAG-PP5, HA-ERK1, or HA-ERK2 were detected following normalization to HSP90 levels. C, HEK-293FT cells were transfected with pcDNA3 (Vector) or FLAG-PP5 in the absence (−) or presence (+) of constitutively active Rac1 (Rac1L61). Cells transfected with Rac1L61 were also treated with 100 ng/ml EGF for 5 min prior to lysis. Endogenous ERK1/2 immune complexes (ERK1/2 IPs) and cell lysates were analyzed by Western using phospho-ERK1/2 (p-ERK1/2), ERK2, PP5, and FLAG antibodies. D, HEK-293FT cells were co-transfected with HA-ERK2 and pcDNA3 (−) or Rac1L61 (+). Western analysis of cell lysates and proteins purifying with normal rabbit IgG (IgG IPs), rabbit anti-PP5 antibody (PP5 IPs), and microcystin-agarose (MC PDs) were performed using ERK2 and PP5 antibodies. E, lysates from untransfected (left) and HA-ERK2-expressing (right) HEK-293FT cells were incubated with microcystin-agarose, and bound proteins were extensively washed prior to splitting the resin into separate tubes, which were then incubated with buffer lacking (−) or containing (+; 20 μg) purified S100A1. A fraction of the reaction mixture was collected and analyzed by SDS-PAGE (15% Tris-glycine gels), and stained with Coomassie G-250 to detect S100A1. Following incubation, bound proteins were extensively washed and eluted for analysis by Western blotting with antibodies detecting the ERK1/2 and PP5 proteins. Unpaired, one-tailed t tests identified a significant decrease in the levels of bound ERK following incubation with S100A1 (left, *, p = 0.0117; right, *, p = 0.0279). Error bars, S.E. The data are representative of experiments performed three (A), three (C), two (D), three (E, left), and two (E, right) independent times with similar results.

Article Snippet: The rabbit anti-PP5 antibody was from Bethyl Laboratories, Inc. (Montgomery, TX).

Techniques: Transfection, Plasmid Preparation, Dominant Negative Mutation, Western Blot, Binding Assay, Expressing, Lysis, Incubation, Purification, SDS Page, Staining, One-tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Small G Proteins Rac1 and Ras Regulate Serine/Threonine Protein Phosphatase 5 (PP5)·Extracellular Signal-Regulated Kinase (ERK) Complexes Involved in the Feedback Regulation of Raf1 *

doi: 10.1074/jbc.M113.518514

Figure Lengend Snippet: Specific oncogenic Ras variants selectively decrease PP5-ERK2, but not PP5-ERK1, interactions. A, HEK-293FT cells were transfected with HA-ERK1, HA-ERK2, or FLAG-PP5 alone or in combination and treated with nothing (Ø), 50 ng/ml EGF (5 min), 100 nm PMA (20 min), or an equivalent volume of DMSO (20 min) as a vehicle control. HEK-293FT cells transfected with FLAG-PP5 and HA-ERK1 or HA-ERK2 in combination with pcDNA3 (Vector) or HRasV12 were not treated prior to lysis. Western analysis of the FLAG immune complexes (FLAG IPs) and cell lysates was done using antibodies recognizing phospho-ERK1/2 (p-ERK1/2), Ras, HSP90, HA, and FLAG. Note that phospho-HA-ERK2 (p-HA-ERK2) and endogenous phospho-ERK1 (endog. p-ERK1) co-migrate. B, HEK-293FT cells transfected with (+) or without (−) HA-ERK2 or FLAG-PP5 were co-transfected with HRasV12, wild type HRas (HRasWT), HA-KRasV12, HA-KRasL61, or wild type HA-KRas (HA-KRasWT). FLAG IPs and cell lysates were analyzed by Western blot as in A. The data are representative of experiments performed four (A) and three (B) independent times with similar results.

Article Snippet: The rabbit anti-PP5 antibody was from Bethyl Laboratories, Inc. (Montgomery, TX).

Techniques: Transfection, Plasmid Preparation, Lysis, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Small G Proteins Rac1 and Ras Regulate Serine/Threonine Protein Phosphatase 5 (PP5)·Extracellular Signal-Regulated Kinase (ERK) Complexes Involved in the Feedback Regulation of Raf1 *

doi: 10.1074/jbc.M113.518514

Figure Lengend Snippet: Kinase activity, but not phosphatase activity, is required for HRasV12-dependent disruption of the PP5·ERK2 complex. A, HEK-293FT cells were transfected with wild type HA-ERK1 or wild type HA-ERK2 together with (+) or without (−) phosphatase-dead FLAG-PP5 (FLAG-PP5PD) and HRasV12. FLAG immune complexes (FLAG IPs) and cell lysates were analyzed by Western blotting using antibodies recognizing the indicated proteins. B, wild type HA-ERK1 (HA-ERK1WT), kinase-dead HA-ERK1 (HA-ERK1KD), wild type HA-ERK2 (HA-ERK2WT), and kinase-dead HA-ERK2 (HA-ERK2KD) were transfected into HEK-293FT cells alone or together with wild type FLAG-PP5 in the absence or presence of HRasV12. FLAG IPs and cell lysates were analyzed as described in A. *, IgG heavy chain. The data are representative of experiments performed three (A) and five (B) independent times with similar results.

Article Snippet: The rabbit anti-PP5 antibody was from Bethyl Laboratories, Inc. (Montgomery, TX).

Techniques: Activity Assay, Transfection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Small G Proteins Rac1 and Ras Regulate Serine/Threonine Protein Phosphatase 5 (PP5)·Extracellular Signal-Regulated Kinase (ERK) Complexes Involved in the Feedback Regulation of Raf1 *

doi: 10.1074/jbc.M113.518514

Figure Lengend Snippet: HRasV12 induces disruption of the PP5·ERK2 complex independently of the activation state of ERK2. A, HEK-293FT cells were transfected with (+) or without (−) HA-ERK2 and FLAG-PP5 together with HRasV12 or wild type HRas (HRasWT); cells were treated with the MEK inhibitor U0126 or DMSO for 30 min prior to lysis. FLAG immune complexes (FLAG IPs) and cell lysates were analyzed by Western using antibodies recognizing the indicated proteins. B, percentage of maximal binding of HA-ERK2 to FLAG-PP5. ERK2 binding signals, quantified for cells in A that co-expressed HA-ERK2 and FLAG-PP5, were normalized to levels of PP5 in the FLAG IPs and compared with the corresponding values in the absence of any Ras expression (Ø), which were set to 100. Significant reductions in PP5 binding to ERK2 were observed in the presence of HRasV12 (86.08 ± 1.62%) and following acute treatment with U0126 (81.95 ± 2.26%), whereas HRasWT expression failed to disrupt the interaction. The results represent the means ± S.E. based on one-way analysis of variance (F(3,8) = 20.42, p = 0.0004). Tukey post-tests are shown as follows: **, versus Ø, p < 0.01; ##, versus HRasWT, p < 0.01. Error bars, S.E. The data are representative of experiments performed three independent times with similar results.

Article Snippet: The rabbit anti-PP5 antibody was from Bethyl Laboratories, Inc. (Montgomery, TX).

Techniques: Activation Assay, Transfection, Lysis, Western Blot, Binding Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Small G Proteins Rac1 and Ras Regulate Serine/Threonine Protein Phosphatase 5 (PP5)·Extracellular Signal-Regulated Kinase (ERK) Complexes Involved in the Feedback Regulation of Raf1 *

doi: 10.1074/jbc.M113.518514

Figure Lengend Snippet: PP5·ERK2 complexes regulate Raf1 feedback phosphorylation, which is elevated in Rac1L61- and HRasV12-expressing cells. A, HEK-293FT cells were co-transfected with Myc-Raf1 or pcDNA3 (EV) together with the indicated combinations of nothing (Ø), wild type (WT) or kinase-dead HA-ERK2 (KD), and wild type (WT) or catalytically inactive FLAG-PP5 (PD). Cells were treated with solvent containing (+) or lacking (−) 100 ng/ml EGF for 30 min prior to lysis. FLAG immune complexes (FLAG IPs) were analyzed by Western using phospho-Ser-289/296/301-Raf1 (pEDS-Raf1), phospho-Ser-338-Raf1 (p338-Raf1), Raf1, HSP90, and FLAG antibodies. B, HEK-293FT cells were co-transfected to express Myc-Raf1, in the presence or absence of Rac1L61 or HRasV12, together with the indicated combinations of nothing (Ø), wild type (WT) or kinase-dead HA-ERK2 (KD), and wild type (WT) or catalytically inactive FLAG-PP5 (PD). FLAG IPs were analyzed as in A. The data are representative of experiments performed two (A) and two (B) independent times with similar results.

Article Snippet: The rabbit anti-PP5 antibody was from Bethyl Laboratories, Inc. (Montgomery, TX).

Techniques: Expressing, Transfection, Lysis, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Small G Proteins Rac1 and Ras Regulate Serine/Threonine Protein Phosphatase 5 (PP5)·Extracellular Signal-Regulated Kinase (ERK) Complexes Involved in the Feedback Regulation of Raf1 *

doi: 10.1074/jbc.M113.518514

Figure Lengend Snippet: A model depicting the role and regulation of PP5·ERK complexes. PP5 suppresses Raf1 signaling by dephosphorylating Ser-338, a site important in making Raf1 permissive to further phosphorylation for full activation. Our studies support an additional role for PP5 in regulating the phosphorylation state of several EDS on Raf1 and suggest that PP5·ERK complexes coordinate Raf1 feedback phosphorylation events. Furthermore, we find that PP5-ERK interactions are modulated by active small G proteins. Active Rac1 (Rac1L61) promotes PP5-ERK1/2 interactions. In contrast, active HRas (HRasV12) and KRas4B (KRasL61), but not KRasV12, promote rapid turnover of PP5·ERK2 complexes without affecting PP5·ERK1 complexes.

Article Snippet: The rabbit anti-PP5 antibody was from Bethyl Laboratories, Inc. (Montgomery, TX).

Techniques: Activation Assay

Journal: Journal of cell science

Article Title: Vimentin and the K-Ras-induced actin-binding protein control inositol-(1,4,5)-trisphosphate receptor redistribution during MDCK cell differentiation.

doi: 10.1242/jcs.108738

Figure Lengend Snippet: Fig. 2. KRAP binds to InsP3R1–GFP and InsP3R3 but not to the deletion mutant. (A,B) Total cell lysates prepared from confluent MDCK cells expressing InsP3R1–GFP or DRD–GFP were incubated with GFP-trap beads. The immunoprecipitated proteins were fractionated by SDS-PAGE, and immunoblotted with antibodies specific for GFP, InsP3R3 or KRAP. (C) Confluent or sparse wild-type MDCK cells were lysed. InsP3R3 was immunoprecipitated and associated KRAP was detected by western blot. (D,E) Protein extracts, prepared from wild-type (wt) DT40 cells or from DT40 triple InsP3 receptor-knockout (TKO) cells, were subjected to SDS PAGE and western blotted using anti-InsP3R1, anti-InsP3R3 or anti-KRAP antibodies. Bands corresponding to full length or truncated forms of InsP3Rs are identified. (F) InsP3R1–GFP was expressed in DT40 TKO cells and precipitated from total cell extracts using GFP-trap beads. The input and bead-bound proteins were separated by SDS-PAGE and analyzed by western blot. The blot membrane displayed in the middle panel was probed with anti-GFP antibodies and then, after stripping, with anti-InsP3R3 antibodies. Non-transfected DT40 TKO cells were used as negative controls. The results shown are representative of two to four independent experiments. IP, immunoprecipitation; NT, non-transfected; WB, western blot.

Article Snippet: The polyclonal antibodies used were all raised in rabbit and directed against actin (Sigma-Aldrich), calnexin (Sigma-Aldrich), erlin-2 (Sigma-Aldrich), KRAP (Proteintech) and InsP3R1 (Parys et al., 1995).

Techniques: Mutagenesis, Expressing, Incubation, Immunoprecipitation, SDS Page, Western Blot, Knock-Out, Membrane, Stripping Membranes, Transfection

Journal: Journal of cell science

Article Title: Vimentin and the K-Ras-induced actin-binding protein control inositol-(1,4,5)-trisphosphate receptor redistribution during MDCK cell differentiation.

doi: 10.1242/jcs.108738

Figure Lengend Snippet: Fig. 3. siRNA-mediated knockdown of KRAP and its effect on InsP3R protein level and localization. MDCK cells expressing InsP3R1–GFP were transfected, using lipofectamine, with different KRAP-targeting siRNAs (referred to as K1, K2 and K3) or with a nonsense duplex (negative control, NC). Cells were processed for western blot or immunofluorescence analysis 72 h post- transfection. (A) Representative western blots showing expression of KRAP, InsP3R1–GFP, InsP3R3 or calnexin (which served as a loading control) in total cell extracts prepared from the siRNA-treated cells. (B) Densitometric analysis of western blots performed with NIH ImageJ software. Data are presented as the means6s.d. of results from three independent experiments. (C) Individual x–y confocal sections showing the distributions of KRAP and InsP3R1–GFP in the siRNA-treated cells. ‘Zooms’ are magnifications of the boxed areas in the adjacent images. Scale bars: 10 mm. The data shown in C are representative of three independent experiments.

Article Snippet: The polyclonal antibodies used were all raised in rabbit and directed against actin (Sigma-Aldrich), calnexin (Sigma-Aldrich), erlin-2 (Sigma-Aldrich), KRAP (Proteintech) and InsP3R1 (Parys et al., 1995).

Techniques: Knockdown, Expressing, Transfection, Negative Control, Western Blot, Immunofluorescence, Control, Software

Journal: Journal of cell science

Article Title: Vimentin and the K-Ras-induced actin-binding protein control inositol-(1,4,5)-trisphosphate receptor redistribution during MDCK cell differentiation.

doi: 10.1242/jcs.108738

Figure Lengend Snippet: Fig. 4. KRAP-binding renders InsP3Rs insoluble in Triton X-100. (A,B) Confluent MDCK cells expressing InsP3R1–GFP or DRD–GFP were lysed by osmotic shock and sequentially extracted with Triton X-100 and SDS to partition cellular proteins into hydro-soluble (HS), Triton X-100-soluble (TS) and Triton X-100-insoluble (TI) fractions. Aliquots of the TS and TI fractions (30 mg of protein) were then resolved by SDS-PAGE, blotted and probed with antibodies recognizing GFP, InsP3R3, KRAP, actin, vimentin or keratins. (A) Western blots representative of the relative distribution of full length and truncated InsP3Rs between the TS and the TI fractions in confluent cells. (B) Quantitative densitometric analysis of western blots from two independent experiments performed with NIH ImageJ software. The results shown are mean6s.d. and take into account both the densitometric value obtained from the immunostaining and the percentage of each fraction loaded. (C,D) TS and TI fractions were prepared from InsP3R1–GFP-expressing MDCK cells transfected, 72 h prior to lysis, with a KRAP-specific siRNA (K2 or K3) or with a control duplex (NC). Samples representing equivalent amounts of proteins from each fraction were subjected to SDS-PAGE, blotted and analyzed for GFP, InsP3R3, KRAP, actin and vimentin. (C) Western blots showing the effect of KRAP depletion on the distribution of InsP3Rs between the TS and TI fractions. (D) Quantitative densitometric analysis of western blots from two independent experiments carried out with NIH ImageJ software. The results shown are mean6s.d. WB, western blot.

Article Snippet: The polyclonal antibodies used were all raised in rabbit and directed against actin (Sigma-Aldrich), calnexin (Sigma-Aldrich), erlin-2 (Sigma-Aldrich), KRAP (Proteintech) and InsP3R1 (Parys et al., 1995).

Techniques: Binding Assay, Expressing, SDS Page, Western Blot, Software, Immunostaining, Transfection, Lysis, Control

Journal: Journal of cell science

Article Title: Vimentin and the K-Ras-induced actin-binding protein control inositol-(1,4,5)-trisphosphate receptor redistribution during MDCK cell differentiation.

doi: 10.1242/jcs.108738

Figure Lengend Snippet: Fig. 5. InsP3R–KRAP complexes are linked to vimentin intermediate filaments. (A,B) Triton X-100-insoluble fractions, isolated from confluent parental MDCK cells (NT) or from cells expressing either InsP3R1–GFP or DRD–GFP, were resuspended in buffer B (containing 1% Triton X-100, 0.5% sodium deoxycholate and 0.1% SDS) and incubated with GFP-trap beads. The input and precipitated proteins were then fractionated by SDS-PAGE, blotted and analyzed with anti-GFP, anti-KRAP, anti-actin, anti-vimentin or anti-keratin antibodies. (C) DT40 TKO cells were lysed in buffer B. Whole-cell lysates were immunoprecipitated with anti-KRAP or control (rabbit IgG) antibodies coupled to protein-G–Sepharose beads. Western blot analysis was performed using anti- KRAP, anti-vimentin and anti-InsP3R3 antibodies. All the western blots shown are representative of three independent experiments. WB, western blot.

Article Snippet: The polyclonal antibodies used were all raised in rabbit and directed against actin (Sigma-Aldrich), calnexin (Sigma-Aldrich), erlin-2 (Sigma-Aldrich), KRAP (Proteintech) and InsP3R1 (Parys et al., 1995).

Techniques: Isolation, Expressing, Incubation, SDS Page, Immunoprecipitation, Control, Western Blot

Journal: Journal of cell science

Article Title: Vimentin and the K-Ras-induced actin-binding protein control inositol-(1,4,5)-trisphosphate receptor redistribution during MDCK cell differentiation.

doi: 10.1242/jcs.108738

Figure Lengend Snippet: Fig. 6. Effects of vimentin knockdown on InsP3R expression, localization and detergent solubility. MDCK cells expressing InsP3R1–GFP were transfected, using lipofectamine, with different vimentin-targeting siRNAs (referred to as V1, V2 and V3) or with a nonsense duplex (negative control, NC). Cells were processed for western blot analysis, immunofluorescence microscopy or subcellular fractionation 72 h post-transfection. (A) Representative western blots showing expression of vimentin, actin (which served as a loading control), InsP3R1–GFP, InsP3R3 or KRAP in total cell extracts prepared from the siRNA- treated cells. (B) Densitometric analysis of western blots performed with NIH ImageJ software. Data are presented as the means6s.d. of results from three independent experiments. (C) Representative x–y projections of stacks of 40 images collected at 0.2 mm intervals along the z-axis, showing the distribution of InsP3R1–GFP (green), KRAP (gray) and vimentin (red) in cells treated with the indicated siRNAs. Scale bar: 10 mm (n53). (D) Fluorescence intensity profiles of InsP3R1–GFP, KRAP and vimentin along the lines indicated in C and obtained from individual x–y sections. (E) Western blots representative of the relative distribution of KRAP, InsP3R1–GFP and InsP3R3 between the Triton X- 100-soluble (TS) and the Triton X-100-insoluble (TI) fractions in the siRNA-treated cells. (F) Quantitative densitometric analysis of western blots from two to four independent experiments performed with NIH ImageJ software. The results shown are mean6s.d. WB, western blot.

Article Snippet: The polyclonal antibodies used were all raised in rabbit and directed against actin (Sigma-Aldrich), calnexin (Sigma-Aldrich), erlin-2 (Sigma-Aldrich), KRAP (Proteintech) and InsP3R1 (Parys et al., 1995).

Techniques: Knockdown, Expressing, Solubility, Transfection, Negative Control, Western Blot, Immunofluorescence, Microscopy, Fractionation, Control, Software, Fluorescence

Journal: Journal of cell science

Article Title: Vimentin and the K-Ras-induced actin-binding protein control inositol-(1,4,5)-trisphosphate receptor redistribution during MDCK cell differentiation.

doi: 10.1242/jcs.108738

Figure Lengend Snippet: Fig. 7. Stable expression of a dominant negative vimentin mutant decreases peripheral accumulation InsP3R3 in polarized MDCK cells. (A) 7 or 10 days after plating, MDCK cells expressing GFP or the VimDN–GFP mutant were fixed, permeabilized, doubled-stained for vimentin and InsP3R3 and then imaged by confocal microscopy. The rabbit anti-vimentin monoclonal antibody (clone EPR3776) that was used recognizes canine vimentin but does not react with the VimDN–GFP mutant. Representative x–y projections and x–z of stacks of 33 images collected at 0.2 mm intervals along the z-axis are presented. Dotted lines highlight the basal membrane. Scale bars: 10 mm. (B) Confluent MDCK cells expressing GFP or VimDN–GFP were lysed by osmotic shock followed by three cycles of rapid freezing and thawing. Membrane and cytoskeletal proteins (M+C) were pelleted by centrifugation at 100,000 g and solubilized in buffer B containing 1% Triton X-100, 0.5% sodium deoxycholate and 0.1% SDS. Hydro-soluble (HS) proteins were recovered in the 100,000 g supernatant. Aliquots of the HS and M+C fractions (30 mg per lane) were analyzed by western blotting using anti-GFP antibodies. The data presented are representative of two independent experiments. (C) (M+C) fractions, isolated from confluent MDCK cells expressing GFP or VimDN–GFP were solubilized in buffer B and incubated with GFP-trap beads. The input and precipitated proteins were then fractionated by SDS-PAGE, blotted and analyzed with anti-GFP, anti-KRAP and anti-InsP3R3 antibodies. The western blots shown are representative of three independent experiments. WB: western blot.

Article Snippet: The polyclonal antibodies used were all raised in rabbit and directed against actin (Sigma-Aldrich), calnexin (Sigma-Aldrich), erlin-2 (Sigma-Aldrich), KRAP (Proteintech) and InsP3R1 (Parys et al., 1995).

Techniques: Expressing, Dominant Negative Mutation, Mutagenesis, Staining, Confocal Microscopy, Membrane, Centrifugation, Western Blot, Isolation, Incubation, SDS Page

Journal: Journal of cell science

Article Title: Vimentin and the K-Ras-induced actin-binding protein control inositol-(1,4,5)-trisphosphate receptor redistribution during MDCK cell differentiation.

doi: 10.1242/jcs.108738

Figure Lengend Snippet: Fig. 8. KRAP and InsP3Rs co-purify with keratin 8 in MCF-7 cells. (A) Representative western blots showing expression of vimentin and keratin 8 in (M+C) fractions isolated, as described in the legend to Fig. 7, from either confluent MDCK cells or MCF-7 cells. (B) Nomarski and corresponding confocal images of MCF-7 cells immunostained for vimentin or keratins are shown. (C) (M+C) fractions, isolated from MCF-7 cells, were solubilized in a buffer containing 1% Triton X-100, 0.5% sodium deoxycholate and 0.1% SDS and incubated with anti-KRAP or control antibodies coupled to protein-G–Sepharose. The input and precipitated proteins were then fractionated by SDS-PAGE, blotted and analyzed with anti-KRAP, anti-pan InsP3R and anti-pan keratin antibodies. The western blots shown are representative of two independent experiments.

Article Snippet: The polyclonal antibodies used were all raised in rabbit and directed against actin (Sigma-Aldrich), calnexin (Sigma-Aldrich), erlin-2 (Sigma-Aldrich), KRAP (Proteintech) and InsP3R1 (Parys et al., 1995).

Techniques: Western Blot, Expressing, Isolation, Incubation, Control, SDS Page