# Role of Hibernation Promoting Factor
## 4 Materials and Methods
### Strains
To assay the ribosomal proteins of purified ribosomes, aliquots (10 \(\mu\)L) of purified ribosomes were separated by SDS-polyacrylamideing gels (16%) and stained with Coomassieie Brilliant Blue R-250 [52]. For immunoblotting of partially purified ribosomes, 10 \(\mu\)L aliquots of the purified ribosome were separated on SDS-PAGE then wet transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Marlborough, MA, USA) at 350 mA for one h. The membranes were washed, blocked, and incubated with polyclonal primary antibodies in 3% skim milk in TBST (Tris buffered saline, 0.1% Tween) overnight on a rocking platform. The membranes were then washed with TBST four times for 15 min each, then incubated in polyclonal rabbit conjugated Horseradish Peroxidase secondary antibody (Thermo Fisher Scientific, Rockford, IL, USA) in 3% Bovine Serum Albumin in TBST for at least three h. Membranes were then washed and developed. Chemiluminescent detection of x-ray autoradiography film (Santa Cruz Biotechnology, Dallas, TX, USA) was performed with an SRX-101A Medical Film Processor (Konica Minolta Medical & Graphic, Inc., Wayne, MJ, USA). Each immunoblot experiment was performed on at least three independent biological replicates. **Ribosomal protein analysis during _P. aeruginosa_ growth. ** Overnight cultures of _P. aeruginosa_ PAO1, _P. aeruginosa_ Alnpf_, _P. aeruginosa_ Alnpf_ + _lnpf_, and _P. aeruginosa_ Aln_AspoT_ were used to inoculate 25 mL of TSB to achieve an OD\({}_{600}\) of 0.01. Carbenicillin and IPTG were added to the medium for the _P. aeruginosa_ Alnpf_ + _lnpf_ strain. The cultures were incubated at 37 \({}^{\circ}\)C with shaking at 200 rpm. Samples (1 mL) were collected after 0, 2, 4, 6, 8, 10, 12, and 24 h of incubation by centrifugation 7690\(\times\)\(g\) for 3 min at 4 \({}^{\circ}\)C. Cell pellets were lysed by incubation in 100 \(\mu\)L Radio Immunoprecipitation Assay Lysis buffer (RIPA) pH 7.6, containing 10 mM Tris-Base, 1 mM EDTA, 140 mM sodium chloride, 0.1% Sodium deoxycholate (DOC), 0.1% sodium dodecyl sulfate (SDS), and 1% NP-40, at 85 \({}^{\circ}\)C for five min. To degrade nucleic acid, 1 \(\mu\)L of Pierce Universal Nuclease (Pierce Biotechnology, Waltham, MA, USA)was added and samples were incubated at room temperature for 5 min. Cellular protein amounts were then determined using the BCA assay (Pierce Biotechnology, Waltham, MA, USA). The mean and standard deviation of total cellular protein recovered from three biological replicates were plotted using GraphPad Prism v 8.3 (GraphPad, San Diego, CA, USA). For samples collected between 4 and 24 h, 15 \(\upmu\)g of total protein from each sample was separated on 16% SDS-PAGE gels, wet transferred to nitrocellulose membranes, and probed with the antibodies generated against HPF, S13, and L5. Developed x-ray films were imaged and band intensities were quantified using Image Studio Software (LI-COR Biosciences, Lincoln, NE, USA). GraphPad Prism was used to plot the mean and standard error of band intensities from three biological replicates. For these assays, GraphPad Prism was also used to perform 2-way ANOVA with Dunnett's test to correct for multiple testing, when necessary. For all statistical tests, an \(\alpha\) of 0.01 was used, and multiplicity adjusted \(p\) values were reported. **Nutrient starvation assays. ** Starved cultures were prepared as described previously [21]. For all strains and media conditions the starved cultures were prepared to achieve approximately \(2\times 10^{8}\) cells/mL as the initial cell concentration in the starvation cultures. For starvation following growth in TSB, overnight cultures of _P. aeruginosa_ and its mutant derivatives were used to inoculate flasks containing 60 mL TSB. For the _hpf_ complemented strain, overnight cultures were supplemented with 150 \(\upmu\)g/mL carbenicillin and 1 mM IPTG. The 60 mL cultures were incubated at 37 \({}^{\circ}\)C with aeration until the OD\({}_{600}\) reached 1.0 (exponential phase) or 6.0 (stationary phase).