1. Volume 13, Issue 1, Pages 125-135 (January 2004)
Contrasting Functions of Calreticulin and Calnexin in Glycoprotein Folding and ER Quality Control 
Maurizio Molinari, Klara Kristin Eriksson, Verena Calanca, Carmela Galli, Peter Cresswell, Marek Michalak, Ari Helenius 
Molecular Cell 
Volume 13, Issue 1, Pages (January 2004)
DOI: /S (03)

2. Figure 1
Chaperone Content in Calreticulin- and Calnexin-Depleted Cells
(A) The relative amount of calreticulin (Crt), calnexin (Cnx), BiP, and ERp57 in wt MEF and in crt−/− MEF was determined by semiquantitative immunoblots of total cell extracts. The lanes were loaded with 2.5 μg of total protein.
(B) As in (A), but for wt and cnx−/− T lymphoblastoid cells.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2
Folding of Influenza Virus HA in wt and in Calreticulin-Depleted MEF
(A) Lysates of influenza virus-infected wt (wt) and calreticulin-depleted MEF (crt−/− ) were immunoprecipitated with a polyclonal antibody raised to influenza virus proteins. IT1, IT2, and NT (native) are three radioactively labeled oxidation intermediates of HA resolved under nonreducing conditions. NP is the nuclear and M is the matrix protein of influenza virus. Upon reduction, HA migrates initially as a single band (ER). The molecular mass of the protein increases (G) at later chase times because N glycans are modified during transit through the Golgi compartment. The ratio NT/IT1+IT2 over time has been plotted. The position of the 200, 116, 97, and 66 kDa protein markers is shown.
(B) Treatment with trypsin of intact cells results in the selective cleavage of surface-exposed HA. Labeled protein retained intracellularly is inaccessible to trypsin, as shown by the persistence of the NP and M proteins. Also refer to Figure 7E.
(C) Cell lysates were immunoprecipitated with an antibody to calnexin (or to BiP) first, then with an antibody to HA to specifically isolate calnexin (or BiP)-bound HA. The amount of calnexin-bound, HA-derived radioactivity has been plotted.
(D) Detergent extracts were precipitated with an antibody specific for HA trimers (N2 [Copeland et al., 1988]).
(E) Sensitivity of HA to endoH treatment. Res (G) is the endoH-resistant, Golgi form of HA; Sens (ER) is the endoH-sensitive, ER form of HA.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
Folding of SFV, VSV, and Cellular Glycoproteins in wt and in Calreticulin-Depleted MEF
(A) Lysates of wt (wt) and of calreticulin-depleted cells (crt−/− ) infected with SFV were subjected to nonreducing and reducing SDS-PAGE. Only viral proteins are labeled because infection with SFV shuts down host protein synthesis. E1 and E2 are the spike glycoproteins of SFV. p62 is the E2 precursor, cleaved by convertase-like enzymes during transit through the Golgi. C is the capsid protein of SFV. Generation of E2, correlating with arrival of SFV glycoproteins in the Golgi, has been plotted.
(B) Noninfected wt and calreticulin-depleted cells were pulsed and chased for the times shown. Cellular substrates associated with calnexin were isolated from detergent lysates by coimmunoprecipitation with the lectin chaperone. Labeled calnexin (Cnx) as well as selected cellular substrates (Y, U, W, X, Z) are shown with arrows.
(C) G protein has been isolated from lysates of wt and calreticulin-depleted cells infected with VSV using a polyclonal antibody to a linear (8685) and to a conformational epitope (I14) of the G protein. The kinetics of generation of the I14 epitope, correlating with acquirement of the native structure, has been plotted.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4
Folding of Influenza Virus HA in wt and in Calnexin-Depleted T Lymphoblastoid Cells
(A and C) As in Figure 2A but for cells depleted of calnexin.
(B and D) As in Figure 2C but with antibody to calreticulin or BiP.
(E) As in Figure 2E.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
Folding of SFV Glycoproteins in wt and in Calnexin-Depleted T Lymphoblastoid Cells
(A) As in Figure 3A but for cells depleted of calnexin.
(B) Detergent extracts were immunoprecipitated with anti-calnexin (left panel) or anti-calreticulin antibody (right panel). Note that an E1 intermediate of oxidative folding (asterisk) persists in association with calreticulin both in wt and in calnexin-depleted T lymphoblastoid cells.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
Folding and Transport of Influenza Virus HA in Cells Treated with Cst
(A) As in Figure 2A for cells treated with Cst.
(B) As in Figure 2E.
(C) Golgi and ER-containing fractions were isolated with isopycnic density centrifugation in Optiprep gradients and treated with detergent. Labeled HA was isolated with specific antibody, treated with endoH, and analyzed in SDS-PAGE (upper panels). Indirect immunofluorescence with HA-specific antibody was performed on nonpermeabilized cells and mock-treated or incubated with Cst during the infection.
(D) Detergent extracts were immunoprecipitated wih an antibody to BiP and then with an antibody to HA to specifically isolate BiP-bound HA, as in Figures 2C and 4D.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7
Assessment of DTT, MesNA, and Trypsin Sensitivity of Total and Surface-Exposed HA Expressed in the Presence of Cst
(A) Assessment of DTT sensitivity has been performed as described. Most of HA expressed in cells treated with Cst remains DTT sensitive (arrow DTTsens) 30 and 60 min after synthesis. Disulfide-bonded complexes containing HA are disassembled upon DTT treatment.
(B) Only the DTT-resistant fraction of HA expressed in the presence of Cst was precipitated with trimer-specific antibodies.
(C) The reducing agent MesNa is impermeable to cells. Accordingly, disulfide-bonded HA complexes formed in Cst-treated cells are not disassembled upon treatment with MesNa (lanes 3 and 4). A significant fraction of labeled HA shifts to a reduced mobility upon treatment with MesNa only in cells treated during the 60 min chase with Cst (MesNa-sensitive fraction). In the reducing gel the slower mobility of glucosylated HA expressed in cells treated with Cst is better appreciated.
(D) Treatment with trypsin of cell lysates is described in the Experimental Procedures. The major proteolytic fragments derived from HA processing are shown (HA1, HA2, XX, YY). NP is trypsin resistant while M is degraded upon treatment with the protease.
(E) As in Figure 2B for cells treated with Cst.
Molecular Cell  , DOI: ( /S (03) )