Table 4.
Complexes containing GpsB and StkP in non-synchronized exponentially growing cells of unencapsulated derivatives of Spn D39a
| GpsB-L-FLAG3 as bait in co-IP experiments | ||||
| Prey proteins tested |
Primary antibodies in Western blot |
Ratio of prey protein band: GpsB-L-FLAG3 bait to GpsB+ control |
Complex detectedb |
|
| bPBP2b-HA | Anti-HA | 18.3 ± 3.1 (n=2) | + | |
| aPBP2a-HA4 | 4.0 ± 1.2 (n=2) | + | ||
| EzrA-HA | 5.4 ± 1.2 (n=2) | + (+B2H) | ||
 |
StkP-HA | 22.6 ± 2.4 (n=2) | + (+B2H) | |
| StkP | Anti-StkP | 25.7 ±2.4 (n=2) | + (+B2H) | |
| MreC | Anti-MreC | 6.2 ± 0.4 (n=2) | + | |
| aPBP1a-HA | Anti-HA | 1.9 ±0.7 (n=2) | ± | |
| bPBP2x-HA | 1.4 ± 0.0 (n=2) | − | ||
 |
HA-FtsA | 1.0 ± 0.0 (n=2) | − | |
| FtsA | Anti-FtsA | 1.2 ± 0.2 (n=2) | − | |
| DivIVA-Myc | Anti-Myc | 1.8 ± 0.3 (n=2) | ± (+B2H) | |
 |
FtsZ-Myc | 1.3 ± 0.4 (n=3) | − (−B2H) | |
| FtsZ | Anti-FtsZ | 1.3 ± 0.1 (n=2) | − (−B2H) | |
| PhpP | Anti-PhpP | 0.8 ± 0.0 (n=2) | − | |
| StkP-FLAG2 as bait in co-IP experiments | ||||
| Prey proteins tested |
Primary antibodies in Western blot |
Ratio of prey protein band: StkP-FLAG2 bait to StkP+ control |
Complex Detected | |
| bPBP2x-HA | Anti-HA | 3.4 ± 0.3 (n=2) | + | |
| bPBP2b-HA | 8.1 ± 0.3 (n=2) | + | ||
| MreC | Anti-MreC | 8.5 ± 0.1 (n=2) | + | |
| FtsZ | Anti-FtsZ | 1.4 ± 0.4 (n=2) | − | |
| FtsA | Anti-FtsA | 1.6 ± 0.1 (n=2) | − | |
| PhpP | Anti-PhpP | 0.7 ± 0.2 (n=2) | − | |
| EzrA-L-FLAG3 as bait in co-IP experiments | ||||
| Prey proteins | Primary antibody in Western blot |
Ratio of prey protein band: EzrA-L-FLAG3 bait to EzrA+ control |
Complex Detected | |
| FtsZ-Myc | Anti-Myc | 15.01 ± 10.2 (n=2) | + (+B2H) | |
Complexes were detected by pairwise co-IP of the indicated bait and prey proteins from bacterial cells that were crosslinked with paraformaldehyde before lysis as described in Experimental procedures. Detection of prey bands by Western blotting (Fig. 9 and S10–S14) is expressed by the mean ratio (±SEM) of luminescence intensity of the prey band recovered from the co-IP to the bait protein compared to the background area from the control mock co-IP from two or more independent experiments. Brackets indicate testing two different versions of the prey proteins.
+, prey band readily detected; −, prey band not detected; ±, prey band was not visible on blots, or if present, is at the limit of detection by this method; (+B2H), putative direct interaction detected in B2H experiments; (−B2H), direct interaction not detected conclusively in B2H experiment (Fig. S15 and data not shown). B2H assays were performed as described in Experimental procedures.