Journal: eLife

Article Title: R7 photoreceptor axon targeting depends on the relative levels of lost and found expression in R7 and its synaptic partners

doi: 10.7554/eLife.65895

Figure Lengend Snippet:

Article Snippet: The primary antibodies used were mouse anti-Chp (1:50; Developmental Studies Hybridoma Bank [DSHB] 24B10), chicken anti-GFP (1:400; Life Technologies), rat anti-HA (1:50; Roche 3F10), rabbit anti-β galactosidase (1:100, Fisher), rat anti-Ncad (1:50; DSHB), rabbit anti-dsRed (1:500; Takara Bio), guinea pig anti-Loaf (1:400, Proteintech), mouse anti-Cnx99A (1:10, DSHB 6-2-1), mouse anti-Hrs (1:10, DSHB 27–4), mouse anti-Rab7 (1:10, DSHB), rabbit anti-ATP6V1B1 (Vha55; 1:200, Abgent), rabbit anti-Arl8 (1:200; DSHB), rat anti-Elav (1:100, DSHB), mouse anti-Notch (1:10; DSHB C17.9C6), mouse anti-Arm (1:10; DSHB N2 7A1), sheep anti-GFP (1:200, Bio-Rad #4745–1051), rabbit anti-RFP (1:500; MBL International #PM005), rabbit anti-V5 (1:1000; Abcam ab9116), mouse anti-FLAG (1:500, Sigma F3165), mouse anti-Dac (1:40; DSHB mAbdac2-3), rabbit anti-Bsh (1:1800) , and guinea pig anti-Runt (1:600; GenScript).

Techniques: CRISPR, Recombinant, Sequencing, Software

Journal: Experimental & Molecular Medicine

Article Title: Extracellular vesicle-mediated delivery of circp53 suppresses the progression of multiple cancers by activating the CypD/TRAP/HSP90 pathway

doi: 10.1038/s12276-025-01506-0

Figure Lengend Snippet: a , GO analysis of RNA-sequencing data revealed that the circp53–209aa is highly associated with mitochondrial pathways. b , Go analysis of RNA-sequencing data revealed that the circp53–209aa is highly associated with apoptotic process signaling pathways. c , A graphic illustration of circp53–209aa interacting with CypD and releasing CypD from the CypD/TRAP1/HSP90 complex, thereby activating its isomerase activity and inducing mPTP opening. d , A Co-IP assay confirmed the direct interaction between circp53–209aa and CypD in MM cells. e ,Representative confocal images of HA and CypD demonstrate the interaction between circp53–209aa and CypD. f , Statistical analysis of the colocation of circp53-209aa and CypD in H929 and XG1 cells. g , A Co-IP assay showed a weaker interaction between CypD and HSP90 in circp53-OE MM cells compared with Ctrl cells. h , Distribution of calcein in H929 and MM1.S cells. i , Distribution of calcein in XG1 and RPMI 8226 cells. j , IOD quantitative statistics of Calcein in Ctrl and circp53–209aa-OE MM cells. k , ATP levels were significantly decreased in circp53–209aa-OE cells. l , Statistical analysis of ATP levels in different cells ( P < 0.001). m , A model showing the high confidence interval of the CypD protein. n , A model of the circp53–209aa-CypD complex. R175 of circp53–209aa binds to E43, E34 and Q163 of CypD through hydrogen bonds and/or ionic interactions. o , Circp53–209aa-R175 is involved in hydrogen-bond interactions with CypD, and these interactions are disrupted upon mutation of arginine to a histidine, as demonstrated by Co-IP using an HA antibody as bait in circp53-OE cells, followed by WB analysis. p , Circp53–209aa-R175H-OE MM cells were constructed successfully, as shown by WB analysis. q , Circp53–209aa-R175H did not influence the proliferation of MM cells. r , Circp53–209aa interacts with CypD and releases it from the CypD/TRAP1/HSP90 complex, as evidenced by Co-IP. s , Colocalization dynamics of CypD/HSP90 in H929 and MM1.S cells. t , Colocalization dynamics of CypD/HSP90 in XG1 and 8226 cells. u , Statistical analysis of the colocation of CypD and HSP90 in H929 and MM1.S cells. v , Statistical analysis of the colocation of CypD and HSP90 in XG1 and RPMI 8226 cells. w , IOD quantitative statistics of CypD and HSP90 in MM cells. x , Distribution of calcein in circp53-OE and circp53–209aa-R175 MM cells. y , The mPTP was not opened in the circp53–209aa-R175H group by quantitative statistics. The data are presented as mean ± s.d. ( n = 3 cultures for each group, * P < 0.05, ** P < 0.01, *** P < 0.001 and ns, no statistical significance).

Article Snippet: The primary antibodies used in this study were diluted at a ratio of 1:1,000, including p53 (10442-1-AP, ProteinTech Group), hemagglutinin (HA) (51064-2-AP, ProteinTech Group), cyclophilin D (CypD) (12716-1-AP, ProteinTech Group), Alix (2171S, Cell Signaling Technology), CD9 (13174S, Cell Signaling Technology), TRAP1 (92345, Cell Signaling Technology), HSP90 (13171-1-AP, ProteinTech Group), BCL-xL (2762s, Cell Signaling Technology), Bax (2774s, Cell Signaling Technology), poly-ADP-ribose polymerase (PARP) (9542S, Cell Signaling Technology), cleaved-Caspase 3 (9661S, Cell Signaling Technology) and β-actin (4970S, Cell Signaling Technology).

Techniques: RNA Sequencing, Protein-Protein interactions, Activity Assay, Co-Immunoprecipitation Assay, Mutagenesis, Construct

Journal:

Article Title: Control of Intracellular Dynamics of Mammalian Period Proteins by Casein Kinase I ? (CKI?) and CKI? in Cultured Cells

doi: 10.1128/MCB.22.6.1693-1703.2002

Figure Lengend Snippet: CKIɛ and CKIδ bind to and phosphorylate mPer proteins specifically. (A) Luciferase (control), mPer1, mPer2, mPer3, mCry1, mCry2, and mBMAL1 were separately synthesized in vitro by using TNT T7 Coupled Reticulocyte Lysate System (right panel) and then mixed with in vitro-synthesized HA-tagged CKIɛ or CKIɛ(KR) and incubated. Each of these mixtures was subjected to immunoprecipitation with anti-HA antibody (12CA5). After resolution by SDS-PAGE, immunoprecipitates were detected by a Bio-Rad phosphorimager (left panel). (B) Myc-tagged mPer1, mPer2, mPer3, mCry1, mCry2, or mBMAL1 was expressed in COS7 cells. Each of the cell extracts was incubated with GST, GST-fused CKIɛ, or GST-fused CKIɛ(KR) (expressed in E. coli, and purified) and glutathione-Sepharose 4B. The beads were then washed with the incubation buffer. After resolution by SDS-PAGE, proteins were analyzed by immunoblotting with anti-Myc antibody (A-14). (C) Myc-tagged mPer1, mPer2, mPer3, mCry1, mCry2, or mBMAL1 was expressed in COS7 cells. The cell extracts were subjected to immunoprecipitation with anti-Myc antibody (9E10). The washed beads were mixed with a kinase reaction buffer supplemented with GST-fused CKIɛ or GST-fused CKIɛ(KR), as well as [γ-32P]ATP, and incubated for 30 min. After resolution by SDS-PAGE, substrate phosphorylation was detected by a Bio-Rad phosphorimager. (D) Myc-tagged mPer1, mPer2, mPer3, mCry1, mCry2, or mBMAL1 was expressed in COS7 cells, together with HA-tagged CKIɛ, CKIɛΔC, CKIδ, or CKIɛ(KR). The cell lysates were subjected to immunoprecipitation with anti-HA antibody (12CA5). After resolution by SDS-PAGE, immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody (A-14). (E) Myc-tagged mPer1, mPer2, or mPer3 was expressed in COS7 cells, together with or without CKIδ. The cell lysates were subjected to immunoprecipitation with anti-Myc antibody (9E10), and the washed beads were resuspended in phosphatase buffer and incubated with or without (mock) 40 U of purified lambda phosphatase for 30 min. After resolution by SDS-PAGE, the immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody (A-14).

Article Snippet: Anti-Myc monoclonal antibody (9E10) and rabbit anti-Myc polyclonal antibody (A-14) were purchased from Santa Cruz Biotechnology, Inc. Rabbit anti-hemagglutinin (HA) polyclonal antibody was from Clontech.

Techniques: Luciferase, Synthesized, In Vitro, Incubation, Immunoprecipitation, SDS Page, Purification, Western Blot

Journal:

Article Title: Control of Intracellular Dynamics of Mammalian Period Proteins by Casein Kinase I ? (CKI?) and CKI? in Cultured Cells

doi: 10.1128/MCB.22.6.1693-1703.2002

Figure Lengend Snippet: CKI-induced nuclear translocation of mPer3 depends on CKI-mediated phosphorylation and NLS of mPer3. (A) Myc-tagged mPer3 was expressed in COS7 cells, together with or without HA-tagged CKIɛ, CKIδ or CKIɛ(KR). The cells were fixed and incubated with primary antibodies (anti-Myc antibody [9E10] and anti-HA polyclonal antibody) and then with the appropriate secondary antibodies. The cells were examined by using a Zeiss Axiophoto. (B, left panel) Myc-tagged mPer3, mut7, or mutNLS (see the right panel) (0.9 μg of DNA) was expressed in COS7 cells, together with or without HA-tagged CKIɛ, CKIδ, or CKIɛ(KR) (0.1 μg of DNA). After being stained, the cells were classified into three categories in terms of location of Myc-tagged mPer proteins as nucleus (N > C), cytoplasm (N < C), or both nucleus and cytoplasm (N = C). The classifications “N > C” and “N = C” are indicated by dark gray and light gray boxes, respectively. More than 150 cells were examined, and the percentages of N > C and N = C are shown. Experiments were performed twice with similar results. (B, right panel) A PCR-generated mutation was made in a presumable mPer3 NLS (mutNLS) and compared to the wild-type (WT) sequence. WT mPer3 or mPer3mutNLS was expressed in COS7 cells, together with or without CKIɛ. The cell extracts were subjected to immunoblotting with anti-Myc antibody (A-14). (C) The cells were subjected to transfection and kept in a medium containing 1% serum, after the serum shock (50% for 2 h). Myc-tagged mPer3 was expressed in COS7 cells, together with or without CKIɛ. The cells were then lysed (or fixed for staining) 30, 36, 42, and 48 h after the serum shock (time = 0 h). In the left panel, cell extracts were subjected to immunoblotting with anti-Myc antibody (A-14). The electrophoretic retardation of the mPer3 protein bands results from phosphorylation. In the right panel, cells were classified after being stained into three categories with regard to the location of Myc-tagged mPer3: N > C, N < C, or N = C. The N > C and N = C classifications are indicated by dark gray and light gray boxes, respectively. More than 250 cells were examined, and the percentages of the N > C and N = C groups are shown.

Article Snippet: Anti-Myc monoclonal antibody (9E10) and rabbit anti-Myc polyclonal antibody (A-14) were purchased from Santa Cruz Biotechnology, Inc. Rabbit anti-hemagglutinin (HA) polyclonal antibody was from Clontech.

Techniques: Translocation Assay, Incubation, Staining, Generated, Mutagenesis, Sequencing, Western Blot, Transfection

Journal:

Article Title: Control of Intracellular Dynamics of Mammalian Period Proteins by Casein Kinase I ? (CKI?) and CKI? in Cultured Cells

doi: 10.1128/MCB.22.6.1693-1703.2002

Figure Lengend Snippet: In vivo association between mPer1 and CKIɛ. (A and B) Various mammalian cells were lysed in the incubation buffer. After the cell extracts were resolved by SDS-PAGE, they were analyzed by immunoblotting with anti-mPer1(N) antibody or anti-actin antibody (loading control) (A) or with anti-mPer1(C) antibody (B). (C) Myc-tagged mPer1, mPer2, or mPer3 was expressed in COS7 cells, and the cell lysates were subjected to immunoprecipitation with anti-Myc antibody (9E10). These immunoprecipitates were analyzed by immunoblotting with anti-mPer1(C) antibody (upper panel) or anti-Myc antibody (A-14) (lower panel). (D) For coimmunoprecipitation experiments, NIH 3T3 cells were incubated in hypotonic lysis buffer and homogenized. Each of antibodies and protein G-Sepharose beads were added to the resulting supernatant. The beads were then washed with the hypotonic lysis buffer. The immunoprecipitates were analyzed by immunoblotting with anti-CKIɛ antibody. Anti-HA antibody and anti-Myc antibody were used as a nonrelevant antibody control.

Article Snippet: Anti-Myc monoclonal antibody (9E10) and rabbit anti-Myc polyclonal antibody (A-14) were purchased from Santa Cruz Biotechnology, Inc. Rabbit anti-hemagglutinin (HA) polyclonal antibody was from Clontech.

Techniques: In Vivo, Incubation, SDS Page, Western Blot, Immunoprecipitation, Lysis

Journal:

Article Title: Control of Intracellular Dynamics of Mammalian Period Proteins by Casein Kinase I ? (CKI?) and CKI? in Cultured Cells

doi: 10.1128/MCB.22.6.1693-1703.2002

Figure Lengend Snippet: Identification of potential CKIɛ phosphorylation sites on mPer3. (A) Amino acid sequences of the mammalian Per family. Amino acid identities and similarities are indicated by dark gray and light gray boxes, respectively. The conserved serine-threonine residues are indicated by open circles. (B) PCR-generated alanine mutations were made in the mPer3 conserved serine-threonine cluster and compared with the wild-type (WT) mPer3 sequence. (C and D) Wild-type (WT) mPer3 or each of various mutants of mPer3 (0.7 μg of DNA) was expressed in COS7 cells, together with or without CKIɛ (0.3 μg of DNA). The cell extracts were subjected to immunoblotting with anti-Myc antibody (A-14). Equal amounts of proteins were loaded. The electrophoretic retardation of the mPer3 protein bands results from phosphorylation. (E) Myc-tagged mut7 was expressed in COS7 cells, together with or without CKIɛ. The cell lysates were subjected to immunoprecipitation with anti-Myc antibody (9E10) and incubated with or without (mock) purified lambda phosphatase. The immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody (A-14).

Article Snippet: Anti-Myc monoclonal antibody (9E10) and rabbit anti-Myc polyclonal antibody (A-14) were purchased from Santa Cruz Biotechnology, Inc. Rabbit anti-hemagglutinin (HA) polyclonal antibody was from Clontech.

Techniques: Generated, Sequencing, Western Blot, Immunoprecipitation, Incubation, Purification

Journal:

Article Title: Control of Intracellular Dynamics of Mammalian Period Proteins by Casein Kinase I ? (CKI?) and CKI? in Cultured Cells

doi: 10.1128/MCB.22.6.1693-1703.2002

Figure Lengend Snippet: CKI promotes ubiquitination of mPer proteins. (A) To show ubiquitination of mPer proteins, each of Myc-tagged mPer proteins was coexpressed with HA-tagged ubiquitin in the presence or absence of CKIɛ, CKIδ, or CKIɛ(KR). The cell lysates were then subjected to immunoprecipitation with anti-Myc antibody (9E10). After resolution by SDS-PAGE, immunoprecipitates were analyzed by immunoblotting with anti-HA polyclonal antibody. The accumulation of high-molecular-mass species recognized by the anti-HA antibody indicates that Myc-tagged proteins become multiply ubiquitinated. (B) To study the metabolic stability of mPer proteins, each of Myc-tagged mPer proteins was expressed in COS7 cells, together with or without CKIɛ. The cells were incubated in methionine-cysteine-deficient medium and then pulsed with 35S-labeled methionine-cysteine. The cells were incubated for the indicated lengths of time in DMEM-10% fetal calf serum with or without MG-132. The cell lysates were subjected to immunoprecipitation with anti-Myc antibody (9E10). The immunoprecipitates were detected with a Bio-Rad phosphorimager.

Article Snippet: Anti-Myc monoclonal antibody (9E10) and rabbit anti-Myc polyclonal antibody (A-14) were purchased from Santa Cruz Biotechnology, Inc. Rabbit anti-hemagglutinin (HA) polyclonal antibody was from Clontech.

Techniques: Immunoprecipitation, SDS Page, Western Blot, Incubation, Labeling