Protein samples were prepared as described in Native PAGE section above, mixed with 2x Laemmli buffer, and boiled for 5 min. Twenty μg of whole cell lysates or 1 μg of affinity-purified chaperone-bound complexes was loaded onto 10% Bis-Tris SDS-PAGE gels. Electrophoresis was conducted for 1 hr at room temperature.
For immunoblotting, a SDS-PAGE gel, or a native PAGE gel was transferred to a PVDF membrane. The PVDF membrane was incubated in 20 mL blocking buffer (TBST; Tris-buffered saline containing 0.1% Tween-20) containing 5% non-fat dry milk, for 1 hr. The membrane was washed twice for 10 min each using TBST. Primary antibodies were diluted in blocking buffer, and were incubated with the membrane for 1 hr, followed by two washes using TBST as above. The HRP-conjugated secondary antibody (1:3000 dilutions in blocking buffer) was incubated with the membrane for 1 hr, followed by two washes using TBST. PVDF membranes were subjected to enhanced chemiluminescence (Perkin Elmer, Western Blot Chemiluminescence Reagents Plus) and were developed using Bio-Rad ChemiDoc MP Imager. Prior to acquiring a ChemiDoc MP Imager, immunoblots were developed using a Kodak X-OMAT processor and X-ray films (Figures 1D , 2F , 3A , and S1A ).
For immunoblotting, a SDS-PAGE gel, or a native PAGE gel was transferred to a PVDF membrane. The PVDF membrane was incubated in 20 mL blocking buffer (TBST; Tris-buffered saline containing 0.1% Tween-20) containing 5% non-fat dry milk, for 1 hr. The membrane was washed twice for 10 min each using TBST. Primary antibodies were diluted in blocking buffer, and were incubated with the membrane for 1 hr, followed by two washes using TBST as above. The HRP-conjugated secondary antibody (1:3000 dilutions in blocking buffer) was incubated with the membrane for 1 hr, followed by two washes using TBST. PVDF membranes were subjected to enhanced chemiluminescence (Perkin Elmer, Western Blot Chemiluminescence Reagents Plus) and were developed using Bio-Rad ChemiDoc MP Imager. Prior to acquiring a ChemiDoc MP Imager, immunoblots were developed using a Kodak X-OMAT processor and X-ray films (