Journal: bioRxiv

Article Title: KaiC-like proteins improve stress resistance in environmental Pseudomonas species

doi: 10.1101/2022.01.26.477890

Figure Lengend Snippet: Aliquots (2.5 µg) of the nickel agarose preparations of P. putida KT2440 KaiC (His-Smt3_KaiC), or of the strep sepharose resin of P. putida histidine kinase (Step_HK) and its PAS domain (Strep_PAS_HK) were analyzed by SDS-PAGE. Coomassie Blue-stained gel is shown. The positions and sizes (in kDa) of marker proteins are indicated on the left.

Article Snippet: Cells equivalent to 1 ml OD600=0.3 were collected; the pellet was resuspended into 300 µl of SDS-PAGE lysis/loading buffer (Wako Chemicals).

Techniques: SDS Page, Staining, Marker

Journal: bioRxiv

Article Title: KaiC-like proteins improve stress resistance in environmental Pseudomonas species

doi: 10.1101/2022.01.26.477890

Figure Lengend Snippet: A. The kaiC gene is maximally transcribed at the transition from exponential phase to idiophase and at the transition from idiophase to stationary phase in P. putida KT2440. B. The kaiC gene is maximally transcribed at the transition from exponential phase to idiophase and at the transition from idiophase to stationary phase in P. protegens CHA0. C. The cellular concentration of FLAG-tagged KaiC, as detected by immunoblot after SDS-PAGE, is minimal in exponential phase (Exp.), but increases dramatically between early stationary phase (ESt., 12h) and mid-stationary phase (MSt., 24h) in P. putida KT2440 and P. protegens CHA0. Two phosphorylation states of the KaiC homolog could be observed in P. putida KT2440 from exponential phase on, while three phosphorylation states were detected in P. protegens CHA0 as of mid-stationary phase.

Article Snippet: Cells equivalent to 1 ml OD600=0.3 were collected; the pellet was resuspended into 300 µl of SDS-PAGE lysis/loading buffer (Wako Chemicals).

Techniques: Concentration Assay, Western Blot, SDS Page

Journal:

Article Title: IL-1? differentially regulates calcium wave propagation between primary human fetal astrocytes via pathways involving P2 receptors and gap junction channels

doi:

Figure Lengend Snippet: Effect of IL-1β on Cx43 expression. (A) Immunoblot analysis of Cx43 protein expression. Membrane extracts were prepared from control cultures (C) and cultures that had been treated with IL-1β for 4 or 24 hr. Five-and ten-microgram samples were loaded and subjected to SDS/PAGE and immunoblot analysis performed for Cx43. Note that of the three forms (NP, P1, and P2) detected, P2 predominated, and that treatment with IL-1β resulted in loss of all three forms from the membrane. Data are representative of four independent experiments. (B) Northern blot analysis of Cx43 mRNA expression in astrocytes. Total RNA was extracted from untreated cultures (C) and cultures treated with IL-1β for the times indicated, separated by electrophoresis, and hybridized with a 32P-labeled probe specific for Cx43. The blots were then stripped and reprobed for 18S rRNA. Note that treatment with IL-1β led to progressive down-regulation of the Cx43 signal. Data are representative of four independent experiments.

Article Snippet: Astrocytes grown to confluence in 100-mm tissue culture dishes (Falcon) and treated as described in Fig. A were lysed in 200 μl of 10 mM Tris⋅HCl hypotonic lysis buffer (4°C, pH 7.5), sonicated, and centrifuged (10,000 × g , 10 min), and the pellets were resuspended in 2× SDS/PAGE loading buffer and separated by using 12% acrylamide gels, followed by Western blotting onto poly(vinylidene difluoride) membrane (Bio-Rad).

Techniques: Expressing, Western Blot, SDS Page, Northern Blot, Electrophoresis, Labeling