Journal:

Article Title: Most F508del-CFTR Is Targeted to Degradation at an Early Folding Checkpoint and Independently of Calnexin

doi: 10.1128/MCB.25.12.5242-5252.2005

Figure Lengend Snippet: Detection of calnexin levels by Western blotting after transient transfection. CHO cells stably expressing either wt CFTR (A) or F508del-CFTR (B) were transiently transfected with human calnexin cDNA (see Materials and Methods). Lanes 1, nontransfected cells; lanes 2, mock-transfected cells (with the empty vector); lanes 3, cells analyzed 24 h posttransfection with calnexin cDNA. Blots were probed with an Ab specific for human calnexin, as indicated on the left.

Article Snippet: The following Abs were used: mouse monoclonal anti-CFTR M3A7 Ab, generated against CFTR amino acids 1197 to 1480 (Chemicon, Temecula, CA; MAB3480); rabbit polyclonal anti-CFTR Ab, generated against a glutathione S -transferase fusion protein containing CFTR residues 1 to 79; monoclonal anti-calnexin AF8 antibody ( 18 ); polyclonal anti-calnexin carboxyl terminus (StressGen, Victoria, BC, Canada; SPA-860); and polyclonal anti-EDEM ER1 and ER2 antibodies ( 35 ).

Techniques: Western Blot, Transfection, Stable Transfection, Expressing, Plasmid Preparation

Journal:

Article Title: Most F508del-CFTR Is Targeted to Degradation at an Early Folding Checkpoint and Independently of Calnexin

doi: 10.1128/MCB.25.12.5242-5252.2005

Figure Lengend Snippet: Turnover and processing of wt and F508del-CFTR under calnexin overexpression. CHO cells stably expressing (A) wt or (B) F508del-CFTR were transiently transfected with the calnexin cDNA construct (lanes 7 to 12) or with the same amount of empty vector as a control (lanes 1 to 6). Twenty-four hours posttransfection, the cells were pulse-labeled for 30 min with [35S]methionine and chased for 0 h (lanes 1 and 7), 0.5 h (lanes 2 and 8), 1 h (lanes 3 and 9), 1.5 h (lanes 4 and 10), 2 h (lanes 5 and 11), and 3 h (lanes 6 and 12). The cells were then lysed and immunoprecipitated with an anti-CFTR Ab (see Materials and Methods). Following electrophoretic separation and fluorography, immature (band B) and mature (band C) forms of CFTR were quantified (see Materials and Methods). Turnover of the core-glycosylated form (band B) of wt CFTR (C) and F508del-CFTR (D) is shown as the ratio between P, the amount of band B at time t, and P0, the amount of band B at the start of the chase (i.e., at the end of pulse). The efficiency of conversion of the core-glycosylated form (band B) into the fully glycosylated form of wt CFTR (band C) was also estimated for wt CFTR (E) and was determined as the ratio between the amount of band C at time t and the amount of band B at the start of the chase (P0). The number of experiments is indicated at the right upper corner of panels C, D, and E. Statistically significant differences are indicated (P < 0.05). (F) (Top) Calnexin cDNA was used to transfect cells stably expressing wt or F508del-CFTR. After being labeled with [35S]methionine, the cells were lysed and CFTR immunoprecipitated with an anti-CFTR Ab. After elution, a second IP was performed (see Materials and Methods) using a mixture (1:1) of human and hamster anticalnexin Abs. Lanes 1 and 3, cells transfected with empty vector (as a control); lanes 2 and 4, cells transfected with calnexin cDNA. (Bottom) Results of direct IP of calnexin in lysates.

Article Snippet: The following Abs were used: mouse monoclonal anti-CFTR M3A7 Ab, generated against CFTR amino acids 1197 to 1480 (Chemicon, Temecula, CA; MAB3480); rabbit polyclonal anti-CFTR Ab, generated against a glutathione S -transferase fusion protein containing CFTR residues 1 to 79; monoclonal anti-calnexin AF8 antibody ( 18 ); polyclonal anti-calnexin carboxyl terminus (StressGen, Victoria, BC, Canada; SPA-860); and polyclonal anti-EDEM ER1 and ER2 antibodies ( 35 ).

Techniques: Over Expression, Stable Transfection, Expressing, Transfection, Construct, Plasmid Preparation, Labeling, Immunoprecipitation

Journal:

Article Title: Most F508del-CFTR Is Targeted to Degradation at an Early Folding Checkpoint and Independently of Calnexin

doi: 10.1128/MCB.25.12.5242-5252.2005

Figure Lengend Snippet: Immunodetection of calnexin levels after transfection with RNAi duplexes specific for this chaperone. (A) CHO cells stably expressing wt CFTR (lanes 1 to 4) or F508del-CFTR (lanes 5 to 8) were transfected with calnexin RNAi duplexes (see Materials and Methods) and analyzed 24 h afterwards. The cells were lysed, and 30 μg of total protein was loaded onto an SDS-PAGE gel. Western blotting was performed using with an Ab recognizing endogenous (hamster) calnexin. Lanes 1 and 5, nontransfected (NT) cells; lanes 2 to 4 and 6 to 8, cells transfected with different amounts of RNAi duplexes (lanes 2 and 6, 20 pmol; lanes 3 and 7, 60 pmol; and lanes 4 and 8, 120 pmol). The arrow indicates detection of calnexin. (B) Blots were scanned, and densitometry was performed for quantification. The results are shown as a plot of the percentage of calnexin present relative to nontransfected cells.

Article Snippet: The following Abs were used: mouse monoclonal anti-CFTR M3A7 Ab, generated against CFTR amino acids 1197 to 1480 (Chemicon, Temecula, CA; MAB3480); rabbit polyclonal anti-CFTR Ab, generated against a glutathione S -transferase fusion protein containing CFTR residues 1 to 79; monoclonal anti-calnexin AF8 antibody ( 18 ); polyclonal anti-calnexin carboxyl terminus (StressGen, Victoria, BC, Canada; SPA-860); and polyclonal anti-EDEM ER1 and ER2 antibodies ( 35 ).

Techniques: Immunodetection, Transfection, Stable Transfection, Expressing, SDS Page, Western Blot

Journal:

Article Title: Most F508del-CFTR Is Targeted to Degradation at an Early Folding Checkpoint and Independently of Calnexin

doi: 10.1128/MCB.25.12.5242-5252.2005

Figure Lengend Snippet: Turnover and processing of wt and F508del-CFTR under calnexin down-regulation by RNAi. CHO cells stably expressing (A) wt or (B) F508del-CFTR were transfected with 60 pmol of RNAi primers specific for calnexin (lanes 6 to 10) or green fluorescent protein RNAi primers as a negative control (lanes 1 to 5). Twenty-four hours posttransfection, the cells were pulse-labeled and chased as before (Fig. ​(Fig.2)2) for 0 h (lanes 1 and 6), 0.5 h (lanes 2 and 7), 1 h (lanes 3 and 8), 2 h (lanes 4 and 9), and 3 h (lanes 5 and 10). The cells were then lysed, immunoprecipitated with an anti-CFTR Ab, and analyzed as before (see the legend to Fig. ​Fig.2)2) to determine the turnover of immature wt CFTR (C) and F508del-CFTR (D) and the processing efficiency of wt CFTR (E). The number of experiments and statistically significant differences are indicated as in Fig. ​Fig.22.

Article Snippet: The following Abs were used: mouse monoclonal anti-CFTR M3A7 Ab, generated against CFTR amino acids 1197 to 1480 (Chemicon, Temecula, CA; MAB3480); rabbit polyclonal anti-CFTR Ab, generated against a glutathione S -transferase fusion protein containing CFTR residues 1 to 79; monoclonal anti-calnexin AF8 antibody ( 18 ); polyclonal anti-calnexin carboxyl terminus (StressGen, Victoria, BC, Canada; SPA-860); and polyclonal anti-EDEM ER1 and ER2 antibodies ( 35 ).

Techniques: Stable Transfection, Expressing, Transfection, Negative Control, Labeling, Immunoprecipitation

Journal:

Article Title: Most F508del-CFTR Is Targeted to Degradation at an Early Folding Checkpoint and Independently of Calnexin

doi: 10.1128/MCB.25.12.5242-5252.2005

Figure Lengend Snippet: Presence of EDEM in CFTR complexes. (A) EDEM cDNA was used alone or with calnexin (CNX) cDNAs to transfect cells stably expressing wt or F508del-CFTR. After being labeled with [35S]methionine, the cells were lysed and CFTR IP was performed with an anti-CFTR Ab. After elution, a second IP was performed using a specific anti-EDEM Ab. Lanes 1 and 4, cells transfected with empty vector (as a control); lanes 2 and 5, cells transfected with EDEM cDNA; lanes 3 and 6, cells cotransfected with EDEM and calnexin cDNAs. As controls, direct IPs of either CFTR (B) or EDEM (C) were also performed after transient transfection with EDEM cDNA.

Article Snippet: The following Abs were used: mouse monoclonal anti-CFTR M3A7 Ab, generated against CFTR amino acids 1197 to 1480 (Chemicon, Temecula, CA; MAB3480); rabbit polyclonal anti-CFTR Ab, generated against a glutathione S -transferase fusion protein containing CFTR residues 1 to 79; monoclonal anti-calnexin AF8 antibody ( 18 ); polyclonal anti-calnexin carboxyl terminus (StressGen, Victoria, BC, Canada; SPA-860); and polyclonal anti-EDEM ER1 and ER2 antibodies ( 35 ).

Techniques: Stable Transfection, Expressing, Labeling, Transfection, Plasmid Preparation

Journal:

Article Title: Most F508del-CFTR Is Targeted to Degradation at an Early Folding Checkpoint and Independently of Calnexin

doi: 10.1128/MCB.25.12.5242-5252.2005

Figure Lengend Snippet: Proposed model for the major degradation pathways of F508del- and wt CFTR. (i) Synthesis of CFTR occurs with its concomitant insertion in the ER membrane and attachment of an Hsc70/Hdj-2 (or Hsp70/Hdj-1) pair to nascent cytosolic domains, as described previously (12, 30). Other authors have described increased levels of Hsc70/Hdj-2 complexes with F508del-CFTR relative to wt CFTR and expression of NBD1 as the earliest point at which Hsc70/Hdj-2 could bind the nascent CFTR polypeptidic chain (30). The same study reported that complex formation between Hdj-2 and nascent wt CFTR was greatly reduced after expression of the R domain, suggesting NBD1-R domain interaction as a critical point in CFTR folding. The cell thus seems to use this Hsc70/Hsp70 control as the first checkpoint to assess CFTR conformation, and we propose that it is the major mechanism to discard F508del-CFTR. Prolonged retention of unfolded F508del-CFTR by Hsc70 at this point enables CHIP to interact with Hsc70/Hsp70 (probably by displacing Hdj-2) and causes the mutant to be degraded through the Hsc70-CHIP-UbcH5a pathway (31, 57). The E2 Ubc6 may also contribute to F508del-CFTR ERAD (24). Contrary to what happens with F508del-CFTR, wt CFTR, for which NBD1-R intramolecular interaction and folding is achieved, proceeds in the folding pathway through interaction of its N-glycosyl residues (ii) with calnexin (iii). Wt CFTR acquires its native conformation through successive rounds of release-deglucosylation (iv) and rebinding-reglucosylation (v) to calnexin, which also constitutes the second ERQC checkpoint. Upon successful folding, CFTR exits the ER, proceeding through the secretory pathway (vi). However, prolonged presence in the calnexin cycle may cause misfolded CFTR to become a substrate of mannosidase I (vii). This enzyme trims mannose residues from the protein glycan moiety, possibly generating the Man8B glycan intermediate that is recognized by EDEM, which targets the client protein to ERAD (viii). We call this ER-degradative pathway GERAD, to indicate its dependence on the glycan moiety. According to this model, F508del-CFTR follows a major degradative pathway from the first (Hsc70-dependent) ERQC checkpoint, whereas misfolded wt CFTR undergoes proteolytic GERAD at the second (calnexin-dependent) one (see the text for a description).

Article Snippet: The following Abs were used: mouse monoclonal anti-CFTR M3A7 Ab, generated against CFTR amino acids 1197 to 1480 (Chemicon, Temecula, CA; MAB3480); rabbit polyclonal anti-CFTR Ab, generated against a glutathione S -transferase fusion protein containing CFTR residues 1 to 79; monoclonal anti-calnexin AF8 antibody ( 18 ); polyclonal anti-calnexin carboxyl terminus (StressGen, Victoria, BC, Canada; SPA-860); and polyclonal anti-EDEM ER1 and ER2 antibodies ( 35 ).

Techniques: Expressing, Mutagenesis

Journal: Scientific Reports

Article Title: Trehalose prevents aggregation of exosomes and cryodamage

doi: 10.1038/srep36162

Figure Lengend Snippet: ( A ) Particle size distribution of beta-ELVs measured by nanoparticle tracking analysis. The size distribution curve fits average particle size and concentration. Data from one representative sample out of twelve are shown. ( B ) Western blot analysis of 15 μg of total cell lysates (MIN6) or beta-ELV proteins reveal the presence of canonical exosomal protein markers and the absence of cellular calnexin. ( C , D ) Experion automated electrophoresis analysis of ( C ) protein profiles under reducing conditions, and ( D ) total RNA isolated from MIN6 cells (upper panel) and of beta-ELVs in PBS or TRE (lower panel). ( E ) Cryo-electronic tomography images of fully hydrated, unstained beta-ELV isolated in PBS or TRE show a population of membrane vesicles of heterogeneous shape and sizes. Scale bars: 100 nm.

Article Snippet: For blotting with polyclonal rabbit anti-calnexin antibody 1:1000 (Euromedex), proteins were separated under reducing conditions and specific sites were blocked by incubation with TBS 0.1% Tween, 5% milk.

Techniques: Concentration Assay, Western Blot, Electrophoresis, Isolation, Tomography, Membrane